Treatment of disease using inter-alpha inhibitor proteins

ABSTRACT

The invention relates to methods of treatment of medical conditions (e.g., diseases and injuries) in a mammal (e.g., a human), such as hypoxia/ischemia, burns, and viral infections (e.g., influenza, West Nile virus, and Dengue fever), in adults and in children (e.g., neonates) by administering a composition that includes an IAIP.

BACKGROUND OF THE INVENTION

Inter alpha inhibitor proteins (IAIPs) are a family of structurallyrelated proteins found in mammalian plasma in relatively highconcentrations. IAIPs play important roles in inflammation as part ofinnate immunity, wound healing, and cancer metastasis (A1-A3). The majorforms found in human plasma are inter-alpha inhibitor (IaI), whichconsists of two heavy chains (H1 and H2) and a single light chain, andpre-alpha inhibitor (PaI), which consists of one heavy (H3) and onelight chain. The light chain (bikunin) is known to inhibit severalserine proteases, such as trypsin, human leukocyte elastase, plasmin,and cathepsin G (A1, A4). The liver is the major site of synthesis ofthe heavy and light chains of IAIP (A3, A5). The high levels ofcirculating IAIPs normally found in plasma of adults and newborns, andeven in prematurely born infants, suggest that these proteins areimportant. Moreover, complete absence of IAIPs has not been reported inhumans (A1), suggesting that these proteins have significant functionsin human biology. In premature infants, IAIPs have recently been shownto decrease in association with sepsis and necrotizing enterocolitis(NEC) (A6-A8). In addition, both disorders are associated with increasedincidences of brain damage in premature infants (A9, A10).

The decreased plasma levels found in septic patients and concomitantincreases of IAIP-related fragments in the urine suggest that theseproteins are “consumed” and rapidly cleared from the systemiccirculation during sepsis (A2, A11, A12). Although the physiologicalfunctions of IAIPs remain to be established, current findings suggestthat these molecules are part of innate immunity and play a criticalrole during inflammation. IAIPs have unique immunomodulatory effects byreducing TNF-α during systemic inflammation and augmentinganti-inflammatory IL-10 during sepsis in neonatal rats (A2, A13, A14).The urinary trypsin inhibitor or bikunin has also been suggested to beeffective in inhibiting premature delivery most likely by suppressingcytokines and other inflammatory mediators (A15-A19). In addition,recent observations demonstrate that IAIPs attenuate complementactivation through the classical and alternative pathways, inhibitcomplement-dependent phagocytosis in vitro, and reducecomplement-dependent lung injury in vivo (A20). These functionspotentially provide mechanistic explanations for its beneficial effectsin systemic inflammation and sepsis and suggest that IAIPs could play animportant role in inflammation-related disorders during the perinatalperiod.

The function of the choroid plexus (CP) and its product cerebral spinalfluid (CSF) has been thought of as providing physical protection to thebrain and facilitating the removal of brain metabolites through thedrainage of CSF. However, more recent studies suggest that the choroidplexus-cerebral spinal fluid system plays a much more active role in thedevelopment, homeostasis, and repair of the central nervous system (CNS)(A39-A41). CP is a highly specialized tissue, strategically positionedwithin the ventricles to provide the CNS with a variety of biologicallyactive growth factors that are essential for normal brain development(A40-A42). These factors include a number of neurotrophic and angiogenicfactors, such as transforming growth factor-α and -β superfamily,insulin-like growth factor, and vascular endothelial growth factor(VEGF), (43-52) and chemo repellents, such as semaphoring 3f and slitprotein (A53, A54) that appear to be involved in neurogenesis and axonalguidance during development of CNS, in response to brain injury, andpossibly in the subsequent repair processes. Previous studies reportedthat during development in many species including human prematureinfants, cerebral spinal fluid has very high protein concentrations,which are most likely important for brain development (A55-A58).Therefore, proteins found in cerebral spinal fluid most likely influencebrain development and responses to injury. Although IAIPs are mostlikely immunomodulatory compounds, their levels have not been previouslyreported in CSF in any species during development.

Information is also very limited regarding the distribution of theseIAIP molecules among different organs, including brain. In humans, IAIPswere detected in cerebrum, cerebellum, lungs, kidney, liver, colon,skin, and testes (A22). Information is not available regarding theexpression of IAIPs in the brain or somatic organs during normaldevelopment.

Tissue ischemia, e.g., persistent restriction of blood supply to atissue, can impair tissue function and result in tissue and organdamage. Tissue ischemia in critical organ systems or body parts, forexample, heart, brain, kidneys, skin, limbs, or gastrointestinal tract,contributes significantly to human morbidity and mortality, and thusthere is a continuing need for therapeutic strategies for treating orprotecting the affected tissues.

SUMMARY OF THE INVENTION

The present invention is based, in part, on our discovery that IAIPs canbe administered (e.g., with a pharmaceutically acceptable carrier) toprovide neuroprotection and to treat tissue ischemia (e.g., in thebrain), including tissue ischemia associated with a disorder, trauma ora congenital defect. The tissue ischemia encompassed by the methods ofthe invention can stem from any of a wide range of medical conditionsthat result in the acute, persistent, or recurring restriction of bloodsupply to the tissue, for example, disorders such as peripheral arterydisease, type 1 or type 2 diabetes, atherosclerotic cardiovasculardisease, intermittent claudication (which can manifest as cramping painin the extremities due to inadequate blood supply), critical limbischemic disease, stroke, myocardial infarction, inflammatory boweldisease, and peripheral neuropathy; traumatic injuries such as wounds,burns, lacerations, contusions, bone fractures, infections, or surgicalprocedures; congenital malformations such as hernias, cardiac defectsand gastrointestinal defects. Thus, tissue ischemia can occur in avariety of tissue types including, for example, skeletal muscle, smoothmuscle, cardiac muscle, neuronal tissue (e.g., the brain), skin,mesenchymal tissue, connective tissue, gastrointestinal tissue and bone.

The present invention provides, in a first aspect, methods of treating,reducing, or inhibiting ischemia or a condition resulting from ischemiain a patient in need thereof. These methods include administering to thepatient a composition including inter-alpha inhibitor (IaI) and/orpre-alpha inhibitor (PaI). In some embodiments, the method treats,reduces, or inhibits ischemia. In some embodiments, the method treats,reduces, or inhibits a condition resulting from ischemia. In someembodiments, the ischemia may be ischemia/reperfusion injury, hypoxicischemia, or hypoxic ischemia encephalopathy. In some embodiments, thecondition resulting from ischemia may be selected from cerebral palsy(CP2), mental impairment, brain damage, paralysis, and neurologicalmorbidity. In some embodiments, the condition resulting from ischemiamay be damage or loss of white matter, white matter demylenation,polymorphonuclear neutrophil infiltration, cerebral cortical injury,inflammation, endothelial activation, cell death, neuronal apoptosis,inhibition of growth, inhibition of development, decreased MBP, alteredcellularity of GFAP positive astrocytes, neuronal apoptosis, decreasedinfarct volume, decreased levels of IαIp, increased plasmin activity,increased activity of metalloproteinases, increased levels of caspase-3,increased levels of Parp1, or increased levels of one or more of thecytokines IL-13, TNF-α, INF-α, IL-6, IL-10, INF-γ, and IL-8. In someembodiments, a method of the present invention may reduce the likelihoodor risk of mortality.

In any of the present methods of treating, reducing, or inhibitingischemia or a condition resulting from ischemia, the ischemia may beacute ischemia. The acute ischemia may be recurring. The ischemia may bepersistent. In any of the above embodiments, the methods of the presentinvention may reduce the severity of ischemia or a condition resultingfrom ischemia or delay the onset or progression of ischemia or acondition resulting from ischemia. In certain embodiments, the ischemiamay result from a medical condition, a traumatic injury, or a congenitalmalformation. Preferably, the medical condition may be selected fromperipheral artery disease, type 1 or type 2 diabetes, atheroscleroticcardiovascular disease, intermittent claudication, critical limbischemic disease, stroke, cancer, myocardial infarction, inflammatorybowel disease, carotid occlusion, umbilical cord occlusion, lowbirth-weight, premature birth, pulmonary insufficiency, peripheralneuropathy, and bleeding (hemorrhagic), the traumatic injury may beselected from wound, burn, laceration, contusion, bone fracture,infection, and surgical procedure, and the congenital malformation maybe selected from hernia, cardiac defect, and gastrointestinal defect. Inparticular embodiments, the ischemia may result from ischemichemorrhagic stroke.

In any of the above embodiments, the ischemia may occur in a tissue orcell type selected from skeletal muscle, smooth muscle, cardiac muscle,connective tissue, mesenchymal tissue, gastrointestinal tissue,placenta, liver, heart, kidney, intestine, lung, colon, kidney, bladder,testes, skin, bone, brain, cerebral cortex, choroid plexus, cerebrum,cerebellum, neurons, astrocytes, and meningeal cells. The ischemia maybe brain ischemia. The brain ischemia may be ischemia of neurons,astrocytes, or meningeal cells of the brain. The brain ischemia may beischemia of the cerebral cortex. The brain ischemia may be the result ofa stroke.

In any of the above embodiments, the patient may be at risk ofexperiencing the ischemia or condition resulting from ischemia. Themethods of the present invention may reduce the severity of the ischemiaor condition resulting from ischemia in the patient. The methods of thepresent invention may reduce the likelihood of manifesting, delay theonset of, or delay the progression of, the ischemia or conditionresulting from ischemia in the patient. The patient may be a fetus, aninfant, or an adult. The fetus may be at risk of premature birth, verylow birth-weight, and/or pulmonary insufficiency. The infant may be bornprematurely, born with a very low birth-weight, have or be at risk ofpulmonary insufficiency, and/or have or be at risk of having an immaturevasculature. The patient may have experienced, or be at risk of,ischemia resulting from umbilical cord occlusion. The patient may haveexperienced, or be at risk of, ischemia resulting from carotidocclusion.

In any of the above embodiments, the patient may be human. In any of theabove embodiments, the patient may have experienced ischemia or acondition resulting from ischemia prior to the administration of thecomposition including inter-alpha inhibitor (IaI) and/or pre-alphainhibitor (PaI). In certain embodiments, the patient may have low levelsof brain IαIp. In any of the above embodiments, the composition may beadministered at a dosage of 1 mg/kg body weight to 50 mg/kg body weight.In any of the above embodiments, the composition may be administered ata dosage ranging from 50 mg/dose to 1000 mg/dose. The composition may beadministered every 4 to 120 hours. The composition may include apharmaceutically acceptable excipient, diluent, or carrier. Thecomposition may be a solid. The solid may be a tablet, capsule, orsuppository. The composition may be a liquid. The composition may beformulated for inhalation, insufflation, nebulization, injection, oral,rectal, topical, or intraperitoneal administration, intracerebralinjection, intravenous delivery, intraarterial delivery, or fetalinfusion. In some embodiments, administration of the composition resultsin a decrease in or down-regulation of one or more cytokines. Thecytokines may be pro-inflammatory cytokines. Any of the above cytokinesmay be intravascular cytokines. Any of the above cytokines may beendothelial-derived cytokines. Any of the above cytokines may begenerated during the ischemia or as a result of a condition resultingfrom ischemia.

In any of the above embodiments, administration of the composition mayresult in a decrease in free radicals. In any of the above embodiments,administration of the composition may result in a decrease in TNF-α. Inany of the above embodiments, the half-life of the composition may be 12to 18 hours.

A further aspect of the invention provides methods of providingneuroprotection to a patient in need thereof, the method includingadministering to the patient a composition including inter-alphainhibitor (IaI) and/or pre-alpha inhibitor (PaI).

In another aspect, the invention provides methods of treating a wound ina patient in need thereof, the method including administering to thepatient a composition including inter-alpha inhibitor (IaI) and/orpre-alpha inhibitor (PaI). The wound may be a burn.

In yet another aspect, the invention provides methods of treating orpreventing a viral infection in a patient in need thereof, the methodincluding administering to the patient a composition includinginter-alpha inhibitor (IaI) and/or pre-alpha inhibitor (PaI). The viralinfection may be influenza, Dengue fever, or West Nile fever. The viralinfection may be H1N1 flu or bird flu.

A further aspect of the present invention provides methods of treatingor preventing cancer metastasis in a patient in need thereof, the methodincluding administering to the patient a composition includinginter-alpha inhibitor (IaI) and/or pre-alpha inhibitor (PaI).

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a subject, together witha composition of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing average Fluoro-Jade B (FjB) staining in ratsfollowing sham treatment, placebo treatment, and treatment with IAIP.Significant increase in total FjB labeled cells in vehicle HI animalscompared to sham and IAIP-treated animals is shown. *p<0.05.

FIG. 2 is a graph showing brain weight in rats following sham treatment,placebo treatment, and treatment with IAIP. Significant reduction inbrain weight in vehicle HI animals (n=11, black bar) compared to sham(n=10, white bar) is shown. No significant reduction in IAIP-treated HIanimals is shown.

FIG. 3 is a set of graphs and insets showing IAIP levels in the brain offetal sheep are dramatically reduced 4 hours after ischemic damage.Fetal sheep were exposed to brain ischemia and reperfusion for 4 h, 24 hand 48 h. There was a dramatic decrease in IAIP levels 4 h after brainischemia, suggesting that IAIPs are consumed during ischemia. Repletionof IAIPs in the brain by intravenous treatment could be the mechanism bywhich treatment with IAIPs could reduce brain damage afterhypoxia-ischemia or stroke. Graphs show pre-alpha inhibitor (125 kDa;left graph) and IAI (250 kDa right graph) Inset shows that pre-alphainhibitor (125 kDa; left inset) and IAI (250 kDa right inset) bands showdecreased expression after 30 min. of ischemia and 4 h of reperfusion(1/R); n=5/group, mean±SEM; *p<0.05 vs control. Expression returnedtoward control values at 24 and 48 h after ischemia.

FIGS. 4A-4C are photographs of hematoxylin and eosin and Luxol fast bluestained sections of fetal sheep brain. The hematoxylin and eosin stainsthe cerebral cortical tissue and the Luxol fast blue stains the whitematter. Infants with CP2 have white matter damage. FIG. 4A shows shamoperated normal fetus. The cortex shows normal cerebral cortical tissueand the white matter shows normal white matter with blue staining. FIG.4B shows a fetus exposed to hypoxia ischemia with a thin ribbon likecerebral cortex and a dramatic loss of white matter. FIG. 4C shows ananimal that was treated with IAIPs IV 4 mg/kg 15 min and 24 and 48 hoursafter carotid occlusion. There is remarkable neuroprotection of bothwhite matter and cerebral cortex. This animal was treated the same as Bbut looks similar to the normal animal in A.

FIG. 5 is a graph showing average FjB labeled profiles.

FIG. 6 is a graph showing the pathological score of brain tissue thathas been treated with a control under normal conditions, a placebo underischemic conditions, and with IAIP under ischemic conditions. Thepathological scoring was performed by a pathologist who did not know thetreatment categories of the fetal sheep. The sham operated control sheepis shown by the control bars for the cerebral cortex and white matter.The hypoxic ischemic sheep are shown by the black bars and the hatchedbars are the hypoxic ischemic sheep that were treated with IAIPs afterischemia as described above. Note that there is about a 50% reduction ininjury to the cerebral cortex and white matter. This is importantbecause damage to the cerebral cortex results in mental retardation andto white matter in CP2.

FIG. 7 is a schematic showing the effect of IAIPs on the brain.

FIG. 8 is a photograph of a gel produced by SDS-PAGE, which shows 125and 250 kDa bands in purified IAIPs from sheep serum.

FIGS. 9A and 9B are graphs and insets showing detection of IAIP duringgestation (70% and 90%) and in newborn and adult sheep. FIG. 9A showsexpression of the 125 kDa IAIP and FIG. 9B shows expression of the 250kDa IAIP.

FIG. 10 is a graph showing an increase in the activity of apro-inflammatory cytokine (caspase 3) in the cerebral cortex of sheepbrain in control and I/R-treated animals (after 4, 24, and 48 hours).

FIG. 11 is a set of photographs showing that neuronal and non-neuronalapoptosis in the ovine fetus can be quantified using NeuN is a neuronalmarker, TUNEL staining to show DNA fragmentation (apoptosis), and DAPIstaining to show nuclei.

FIG. 12 is a graph showing that IAIP plasma levels in mice peaked 6 hafter injection (at both 30 mg/kg and 60 mg/kg body weight (bw) anddecreased by 24 h. IAIP concentration was detected using a competitiveELISA assay with monoclonal antibody 69.31 specific against the lightchain of human IAIP.

FIG. 13 As shown in the schema below, after baseline measurements & 30min ischemia, fetal sheep will receive placebo or IAIP (4 mg/kg fetalweight) 15 min, 24 h and 48 h after the onset of reperfusion. Thistreatment regimen was selected because the 8-12 h half-life of IAIPsshould provide fetal exposure to IAIPs for the majority of the 72 hreperfusion, and this dose appears efficacious in our preliminary data.Measurements obtained at baseline, during ischemia, after 30 min ofischemia, and sequentially during reperfusion (study design, solidcircles) include fetal heart rate, mean arterial blood pressure,amniotic fluid pressures, continuous ECoG, and separate sets of bloodcollection. At the end of the study, the ewe and fetus will be givenintravenous pentobarbital (15-20 mg/kg) to achieve a surgical plane ofanesthesia and 200 mg/Kg of pentobarbital for euthanasia. Blood samples(study design, solid circles) are obtained for hematocrit, blood gases,oxygen saturation, arterial glucose and lactate, IAIP concentration, andcytokines (IL1-β and IL-6).⁹⁹

DETAILED DESCRIPTION OF THE INVENTION

We have discovered methods for the treatment of tissue ischemia, inparticular ischemia of the brain, using IAIPs. These methods can beapplied to, and are expected to benefit subjects having any of a varietyof medical conditions that can give rise to tissue ischemia. The methodsare based, inter alia, on the inventor's discovery that administrationof IAIPs or a pharmaceutical composition comprising IAIPs to a subjecthaving or likely to develop tissue ischemia.

Compositions

The pharmaceutically acceptable compositions of the invention includeIAIPs in dosages known in the art (see, e.g., U.S. Pat. No. 7,932,365and US 2009/0190194, each of which is incorporated herein by referencein its entirety).

For example, compositions of the invention can be administered in adosage ranging from about 1 to 50 mg/kg of body weight, preferablydosages between 500 mg and 1000 mg/dose, every 4 to 120 hours, or asneeded.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly 0.8 throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedherein and/or known in the art. In some embodiments, the compositionsare administered by the oral or nasal respiratory route for local orsystemic effect. Compositions in can be nebulized by use of inert gases.Nebulized solutions may be breathed directly from the nebulizing deviceor the nebulizing device can be attached to a face masks tent, orintermittent positive pressure breathing machine. Solution, suspension,or powder compositions can be administered orally or nasally fromdevices which deliver the formulation in an appropriate manner.

The compositions administered to a patient can be in the form of one ormore of the pharmaceutical compositions described above. Thesecompositions can be sterilized by conventional sterilization techniquesor may be sterile filtered. Aqueous solutions can be packaged for use asis, or lyophilized, the lyophilized preparation being combined with asterile aqueous carrier prior to administration. The pH of the compoundpreparations typically will be between about 3 and 11, for example,between about 5 to 9, between 6 and 7, or between 7 and 8. It will beunderstood that use of certain of the foregoing excipients, carriers, orstabilizers will result in the formation of pharmaceutical salts.

The proportion or concentration of a compound of the invention in apharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the compounds of the inventioncan be provided in an aqueous physiological buffer solution containingabout 0.1 to about 10% w/v of the compound for parenteral adminstration.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asdα-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompositions described herein.

The pharmaceutical compositions of this invention may contain anyconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsor vehicles. In some cases, the pH of the formulation may be adjustedwith pharmaceutically acceptable acids, bases or buffers to enhance thestability of the formulated composition or its delivery form. The termparenteral as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques. Suitable methods of administration maybe as a tablet, capsule, or by intravenous injection. Injectable formsof administration are particularly preferred.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a composition of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is useful when the desired treatment involves areas or organsreadily accessible by topical application. For application topically tothe skin, the pharmaceutical composition should be formulated with asuitable ointment containing the active components suspended ordissolved in a carrier. Carriers for topical administration of thecompositions of this invention include, but are not limited to, mineraloil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene composition, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active composition suspended ordissolved in a carrier with suitable emulsifying agents. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. The pharmaceuticalcompositions of this invention may also be topically applied to thelower intestinal tract by rectal suppository formulation or in asuitable enema formulation. Topically-transdermal patches are alsoincluded in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

Treatment of Disease Using IAIPs

Tissue Ischemia

Tissue ischemia is associated with a wide range of medical conditionsthat result in partial, substantially complete or complete reduction ofblood flow to a body part or tissue comprising a body part and may bethe result of disease, injury, or of an unknown cause, and may beinfluenced by one's genetic constitution. Regardless of the medicalcondition leading to tissue ischemia, a patient who has or is likely todevelop tissue ischemia is a candidate for treatment with thepharmaceutically acceptable IAIP compositions described herein.Treatment can completely or partially abolish some or all of the signsand symptoms of tissue ischemia, decrease the severity of the symptoms,delay their onset, or lessen the progression or severity of subsequentlydeveloped symptoms.

IAIPs are important in inflammatory responses during the perinatalperiod as evidenced by our previous findings that they are dramaticallydecreased in response to sepsis and NEC in premature infants (A13,A15-A19), (A7, A8), and are important in ischemic and inflammatoryrelated brain and somatic organ damage in adult rats (A31, A32).Therefore, given the potential importance of these molecules inperinatal period during inflammatory states, and the fact that we haverecently shown that they are present in high levels in the normal brainand in somatic organs during development and that damage is associatedwith decreases in IAIPs in ischemia related disorder in adult subjects,we believe these molecules represent endogenous immuno-protectivemolecules in most organs during development. Furthermore, we hypothesizethat administration of these molecules most likely will prove to havegreat therapeutic potential during the perinatal period and in adultswith stroke.

In addition, data suggests that IAIP plays a role in down-regulation ofsystemic inflammatory cytokines, including a systemic effect measured inthe brain. IAIPs are believed to function in a unique manner at theblood-brain barrier (“BBB”), and have a positive effect on thesuppression of free radicals. IAIP also delivers an additional advantageover bikunin, due to significantly increased half-life. IAIP half-lifeis in the range of 12-18 hours, while bikunin half-life is in the rangeof 3-8 minutes. Therefore, systemically delivered, e.g., IV delivered,IAIP has the benefit of system-wide distribution, and steadytime-release of Light Chain proteins over a significantly increasedtime. In other words, IAIP acts as a transport agent and releasemechanism modulator to more effectively deliver a therapeutic benefitover a longer period of time, and over a broader systemic area. As IAIPseparates into the heavy and light chain components that make up IAIP, acomplementary series of therapeutic benefits ensues, over a longerperiod of time than any previous therapy.

Summary of Benefits of Treatment According to the Invention IAIP as abiomarker for brain injury: IAIP levels in the brain, or “Brain IAIP”decreases markedly after exposure of the brain to hypoxic ischemia. Datashow that Brain IAIP is directly correlated to brain injury due tohypoxic ischemia. Replacing IAIP through IV delivery post hypoxicischemia has the benefit of replacing the IAIP and increasing systemic,IAIP levels and brain IAIP levels. Furthermore, data show that IAIP isan accurate biomarker for brain injury.

IAIP is effective as a neuroprotectant following hypoxic ischemia orstroke. We hypothesize that replacing IAIP through IV delivery afterhypoxic ischemia or stroke will most likely increase systemic IAIPlevels. We have shown that IV administration of IV IAIP to fetal sheepmarkedly decrease brain injury in the perinatal brain (FIG. 4A-4C), andreduces Pathological Scoring of uninjured tissue as measured in CerebralCortex and White Matter by a pathologist who was not aware of thetreatment of the animals (FIG. 6).

IAIP levels are very high in the Cerebral Spinal Fluid (“CSF”) in thefetus, and IAIP levels drop precipitously upon birth. This endogenoushigh level of IAIP present in fetal CSF suggests that IAIPs are criticalin the development of the prenatal brain. Adults do not have any IAIPpresent in their CSF, as measured by Western Blot testing. This earlydata is significant as it underscores the unique and significantcorrelation between IAIP and fetal brain development.

IAIP acts a positive mediator for Ischemic Reperfusion Injury (“IR”). Ithas been shown that hypoxic ischemia and/or stroke lead to cerebralpalsy (CP2), and mental retardation. One understood physiologicalcontributor to this clinical issue is IR. IAIP has been shown to reduceand mitigate the deleterious effects of ischemic IR.

Neuroprotection and Treatment of Hypoxic Ischemia in Neonates

The only approved treatment for neonatal hypoxia-ischemia ishypothermia, which is only partially protective. Additional treatmentsthat provide greater neuroprotection are vitally needed for treatingthis disorder in neonates. Hypoxia-ischemia at birth results in a greatburden lifelong burden to the individual and society. There is only onetreatment for stroke in adult patients and it has a very limited scopeas it must be used within 4.5 hours of the development of stroke. Thereis not treatment for stroke in newborns. This is important as theincidence of stroke in the newborn is the same as in adult patients.

While much of the data has been heretofore focused on animal studies, toinclude sheep studies, it must be noted that the only study measuringefficacious treatment for human infants was performed using the samesheep model that the inventors used.¹

Sepsis and NEC-related decreases in IAIPs could account for the reportedincreased incidence of brain damage in exposed premature infants^(47,48)and IAIPs could represent neuroprotectants in this population. Inaddition, prior to the data described herein and incorporated in itsentirety, IAIPs have not been studied in the immature brain, making theconclusions disclosed herein highly novel. IAIPs are novelanti-inflammatory molecules that robustly block increases inpro-inflammatory cytokines in response to sepsis, and augment the risein anti-inflammatory cytokine production.¹¹

Our data in fetal sheep show that IAIPs have remarkable neuroprotectiveproperties. Based upon our data during the perinatal period in fetalsheep, we believe administration of IAIPs can prevent some elements ofbrain damage in human infants and would be feasible as human bloodproducts are currently used to treat infants. In addition, IAIPs couldalso be an adjunctive treatment to the partial protection afforded byhypothermia in full-term infants.⁸⁷ The IAIPs could also have asignificant translational potential to prevent or attenuate brain damagein infants at risk for mental retardation and CP2. Many of the infantswho are at risk for CP2 are premature infants and there is absolutely nopreventive or therapeutic strategy for these infants except for theadministration of magnesium sulfate to the mother, which has verylimited protective properties only in some infants.

The issues with the current gold standard of treatment with hypothermiaare as follows: For neo-natal patients, the issue is Hypoxic IschemicEncephalopathy (“HIE”) that is currently poorly treated with hypothermiatreatment. The neonatal patient, when diagnosed with hypoxic ischemia,is placed on a cold (32 degrees F.) circulating mattress to cool theirsystem. Treatment does not result in treatment of the symptoms andunderlying issues, but only partially mitigates the impact of HIE.Patients are not returned to a normal healthy state after HIE treated byhypothermia. The current gold standard is only partially effective.Consequently, there is an urgent need to find additional adjunctivetreatments.

The methods of the present invention include the administration of anIAIP to a neonate in need thereof for the treatment of tissue ischemia,such as HIE. The method includes administering an IAIP to a neonate atrisk of ischemia or other brain injury (e.g., as a neuroprotective), aswell as to neonates diagnosed with ischemia or other brain injury.

The present invention is significant in that the frequency and severityof negative outcomes following Neonatal Encephalopathy are excessivelyhigh. Between 40 and 58% of the neonatal patients who experiencedhypoxic ischemia died or had severe mental disability, as measured by IQscore less than 70. No current treatment effectively or satisfactorilyaddresses the primary concerns leading to death or significant mentalimpairment following hypoxic ischemia in neonatal patients.

The following is excerpted from the New England Journal of Medicine,incorporated in its entirety by reference: New England Journal ofMedicine, “Childhood Outcomes after Hypothermia for NeonatalEncephalopathy”, Shankaran et al, 2012; 366:2085-92:

-   -   BACKGROUND We previously reported early results of a randomized        trial of whole-body hypothermia for neonatal hypoxic-ischemic        encephalopathy showing a significant reduction in the rate of        death or moderate or severe disability at 18 to 22 months of        age. Long-term outcomes are now available.    -   METHODS In the original trial, we assigned infants with moderate        or severe encephalopathy to usual care (the control group) or        whole-body cooling to an esophageal temperature of 33.5° C. for        72 hours, followed by slow rewarming (the hypothermia group). We        evaluated cognitive, attention and executive, and visuospatial        function; neurologic outcomes; and physical and psychosocial        health among participants at 6 to 7 years of age. The primary        outcome of the present analyses was death or an IQ score below        70.    -   RESULTS Of the 208 trial participants, primary outcome data were        available for 190. Of the 97 children in the hypothermia group        and the 93 children in the control group, death or an IQ score        below 70 occurred in 46 (47%) and 58 (62%), respectively        (P=0.06); death occurred in 27 (28%) and 41 (44%) (P=0.04); and        death or severe disability occurred in 38 (41%) and 53 (60%)        (P=0.03). Other outcome data were available for the 122        surviving children, 70 in the hypothermia group and 52 in the        control group. Moderate or severe disability occurred in 24 of        69 children (35%) and 19 of 50 children (38%), respectively        (P=0.87). Attention-executive dysfunction occurred in 4% and        13%, respectively, of children receiving hypothermia and those        receiving usual care (P=0.19), and visuospatial dysfunction        occurred in 4% and 3% (P=0.80).    -   CONCLUSIONS The rate of the combined end point of death or an IQ        score of less than 70 at 6 to 7 years of age was lower among        children undergoing whole-body hypothermia than among those        undergoing usual care, but the differences were not significant.        However, hypothermia resulted in lower death rates and did not        increase rates of severe disability among survivors. (Funded by        the National Institutes of Health and the Eunice Kennedy Shriver        NICHD Neonatal Research Network; ClinicalTrials.gov number,        NCT00005772.)

Limitations of Treatment by Hypothermia

The following is excerpted from the New England Journal of Medicine,“Whole-Body Hypothermia for Neonates with Hypoxic-IschemicEncephalopathy”, Shankaran et al, 2005; 353:1574-84:

-   -   Background: Hypothermia is protective against brain injury after        asphyxiation in animal models. However, the safety and        effectiveness of hypothermia in term infants with encephalopathy        is uncertain.    -   Methods: We conducted a randomized trial of hypothermia in        infants with a gestational age of at least 36 weeks who were        admitted to the hospital at or before six hours of age with        either severe acidosis or perinatal complications and        resuscitation at birth and who had moderate or severe        encephalopathy. Infants were randomly assigned to usual care        (control group) or whole-body cooling to an esophageal        temperature of 33.5° C. for 72 hours, followed by slow rewarming        (hypothermia group). Neurodevelopmental out-come was assessed at        18 to 22 months of age. The primary outcome was a combined end        point of death or moderate or severe disability.    -   Results: Of 239 eligible infants, 102 were assigned to the        hypothermia group and 106 to the control group. Adverse events        were similar in the two groups during the 72 hours of cooling.        Primary outcome data were available for 205 infants. Death or        moderate or severe disability occurred in 45 of 102 infants (44        percent) in the hypothermia group and 64 of 103 infants (62        percent) in the control group (risk ratio, 0.72; 95 percent        confidence interval, 0.54 to 0.95; P=0.01). Twenty-four infants        (24 percent) in the hypothermia group and 38 (37 percent) in the        control group died (risk ratio, 0.68; 95 percent confidence        interval, 0.44 to 1.05; P=0.08). There was no increase in major        disability among survivors; the rate of cerebral palsy was 15 of        77 (19 percent) in the hypothermia group as compared with 19 of        64 (30 percent) in the control group (risk ratio, 0.68; 95        percent confidence interval, 0.38 to 1.22; P=0.20).    -   Conclusions: Whole-body hypothermia reduces the risk of death or        disability in infants with moderate or severe hypoxic-ischemic        encephalopathy.

Neuroprotection and Treatment of Hypoxic Ischemia in Adults

The same thought process applies to the neuroprotective properties ofIAIP for adult patients who have experienced stroke. For adult patients,stroke is the largest issue, and may lead towards mental impairment andparalysis. stroke is currently treated with a limited clinicalarmamentarium, with Tissue Plasminogen Activator (“TPA”) as the leadtreatment method. tPA must be administered within 4.5 hours of thestroke event, and is not fully effective. tPA is a blood thinning agent,which makes it contraindicated for patients with bleeding issues and forpatients with potential hemorrhagic transformation of stroke.

Adults suffering a stroke are not treated with hypothermia. Instead,stroke patients are treated with Tissue Plasminogen Activator (“tPA”).tPA is a thrombolytic agent used in diseases that feature blood clotsdue to events such as pulmonary embolism, myocardial infarction, andstroke. tPA must be administered as quickly as possible after themedical event in order to be as effective as possible, and is intendedto be administered within 4.5 hours of the event. The efficacy of tPA asa treatment for stroke has not been proven, and remains a source ofcontroversy. See, e.g., Western Journal of Medicine, “Truths about theNINDS Study: Setting the Record Straight”, West J Med. 2002 May; 176(3):192-194, Jeffrey Mann:

-   -   Thrombolysis for acute ischemic stroke has been studied for more        than a decade, but its efficacy remains controversial. The first        study to claim that tissue plasminogen activator (tPA) is        effective in the treatment of acute ischemic stroke was a        multicenter clinical trial coordinated by the National Institute        of Neurological Disorders and Stroke (NINDS) Study Group. The        NINDS study's conclusions, published in 1995,1 were that        “treatment with intravenous tPA within 3 hours of the onset of        ischemic stroke improved clinical outcome at 3 months . . . .        [A]s compared with patients given placebo, patients treated with        tPA were at least 30% more likely to have minimal or no        disability at 3 months.” 1(p 1586) The NINDS study was widely        perceived to be a well-executed and analyzed randomized        controlled trial, and its results were well received by many        medical professionals and the public.”    -   In summary, the recommendations for the use of tPA in patients        with acute ischemic stroke were based on an initial        misinterpretation of the results of the NINDS trial and are,        therefore, unwarranted. The NINDS investigators may think that        tPA works and that no further trials are needed. In fact, Lyden        in an editorial in “Controversies in Stroke” wrote, “Perhaps we        will find a way to treat patients later than 3 hours, and        further studies are needed to push the outer limits of the time        window, but within the 3-hourwindow, no further trials are        needed; the drug works. The dictum primum nonocere still        applies: we must do no harm, either by actively committing an        act or by withholding a proven therapy through inaction.”7(p        2709). The readers of this article should think carefully about        these issues and independently decide whether further trials of        the use of tPA for acute ischemic stroke are needed.        In addition, administering tPA is difficult, impractical, and        does not produce a positive clinical benefit in any more than        10% of the studied patient population when compared to placebo.        See, e.g., Cleveland Clinic Journal of Medicine, volume 69,        number 9, September 2002, “Acute Stroke Therapy: beyond IV tPA”,        Furlan is incorporated in its entirety by reference.    -   tPA FOR STROKE:EFFECTIVE BUT OFTEN IMPRACTICAL In a landmark        study from the National Institute of Neurological Disorders and        Stroke (NINDS),1 624 patients were randomized to receive either        placebo or IV tPA (0.9 mg/kg, maximum 90 mg, 10% as a bolus and        the remainder within 60 minutes) within 3 hours of stroke onset.        At 90 days, there was an 11% to 13% absolute increase in        essentially full neurologic recovery among treated patients. But        at a price. The rate of symptomatic intracerebral hemorrhage at        36 hours was significantly higher in the tPA group (6.4% vs        0.6%). Although overall mortality was not increased, tPA-related        intracerebral hemorrhage is often fatal. The net benefit of tPA        was reduced for older patients (age>77 years) and for more        severely affected patients (ie, with a National Institutes of        Health Stroke Scale [NIHSS] score>22).    -   The Food and Drug Administration (FDA) approved tPA for treating        acute ischemic stroke in June 1996, but only for patients        meeting the inclusion criteria of the NINDS study. Most        important of these: treatment must begin with-in 3 hours of the        onset of stroke, and before this can happen, patients must        undergo a computed tomographic (CT) scan to rule out        intracerebral hemorrhage.    -   Using IV tPA in clinical practice has proved very difficult. For        example, in Cleveland hospitals in 1997-1998, only 1.8% of        patients admitted with ischemic stroke received IV tPA.2        Further underscoring the impracticalities of administering tPA        to combat the degenerative effects of stroke are described by        Kevin Pho, MD, ER Stories, MD in Conditions, Sep. 1, 2010: “The        TPA Time Limit for Stroke Causes Mass Chaos in the ER”:    -   I hate acute strokes. There are several reasons for it. Most of        them are logistical. First, everyone gets into a tizzy because        of the 3 (or 4.5) hour time limit after the onset of symptoms        that which TPA can be given and hopefully improve the patient's        outcome. Unfortunately, this time limit (and the data for TPA's        efficacy is only OK at best) causes mass chaos and annoyance.    -   First, one has to establish 100% what the exact time of onset        was. This is not easy most of the time. I would say about 80% of        “acute” strokes brought in by EMS turn out to not be within that        window. It takes more than just saying “when did the symptoms        start?”    -   Often the patient is elderly and demented. Often they live        alone. Often there were milder symptoms before that were ignored        or unrealized. Occasionally the person has hemi-neglect and        can't really say when things started. Sometimes there is alcohol        on board. Sometimes the symptoms are on top of pre-existing        stroke damage and it is hard to tell if it is really new or        worse. Sometimes patients probably had a seizure at onset and        that prevents them from getting TPA.    -   All these things make history taking a royal pain in the ass.        And remember, it must be done quick! The exam can be hard too.        Sometimes the patient can't reliably follow commands or there is        a language barrier. Sometimes the patient's preexisting abnormal        findings make it hard to tell if something is old or not.        Sometimes the person is so out of it the whole thing is a waste        of time.    -   Second, once you are sure it is a stroke, you have to hustle. If        the person came in within one hour, no prob. But if 2 have        passed (or 3.5 in a younger patient eligible for the 4.5 hour        window), it is tough. The bloods have to be sent off. Blood        pressure may have to be corrected. You have to zoom the patient        over to CT and get it read. You have to get consent (often from        a family member who is on the telephone), as well as the worst        part of all. That would be calling the neurologist.    -   Many hospitals (like mine) require that the giving of TPA is a        two-doctor job—and one is the neurologist. I think mostly        because neurologists are the best at making sure it really is a        stoke. In many cases it is pretty obvious, but in the        borderline, more challenging cases, they are much more astute        than me at teasing out the minutiae from the history and subtle        exam findings. This is important because TPA has a big risk;        bleeding like stink. Turn a ischemic stoke into a hemorrhagic        one and you've screwed the patient royally. Cause a bleed in        someone who really was not having a stoke? You are so screwed it        is not even funny.    -   Anyway, calling the neurologist sucks. Why? The same reason it        sucks for everyone else. They have to drop whatever it is they        are doing and come flying in. As you can guess, strokes that        happen at 2 am are truly unwelcome. They hate to get awoken, and        I hate to wake them. Even if it is during the day, they have to        abandon their rounds or their patients in the office to come in.        Of course I know it sucks (it wrecks my rhythm too) but part of        me is just like, “You guys did the research for this stuff and        published the papers and made it standard of care.”    -   Regardless, one cannot explain how grumpy and unpleasant to deal        with the neurologist is at 4 am. If anything is out of place, if        the flow of things is not perfectly smooth, or if the nurses        don't have everything ready for them, it's freak-out time. God        forbid if the diagnosis is wrong. Or if they feel the symptoms        started earlier and the patient is out of the window. Or if it        turns out the patient has some contraindication to getting the        drug. Lets just say the discussion between the doctors is not        pleasant.    -   All this is bad enough but what really takes the cake is that        the treatment is not very good. The data in the big studies is        sub-par (certainly compared to many other treatments for things        we do). Even under the best of circumstances (which seem to        almost never occur) the improvement the patient gets is only        moderate (and even worse during the 3-4.5 hour window). Of        course, that may be significant in the long run for the        patient's functioning but a good part of the time, they don't        improve at all.    -   Add that in with the people who bleed and you have a treatment        that few people are enthusiastic about. Of course this leads to        another part, the giving or not giving of TPA in acute stroke is        a huge lawsuit waiting to happen. If you give it and the person        does poorly, you get sued. If you don't give it and the person        does worse, you get sued. So, I say please invent something        better for strokes.    -   Finally, of course, I hate it for what it does to patients. It        can be truly devastating and the costs to the patient, family,        and society is staggering.        New device innovation is focused on endovascular cooling devices        vs. external heat-exchange cooling surfaces. Hypothermia is        widely believed to offer the following benefits (Cleveland        Clinic, Furlan):    -   Hypothermia may exert its effect by reducing glutamate release,        free-radical mechanisms. ischemic depolarization, and kinase        reactions; by preserving the blood-brain barrier and        cytoskeleton; and by suppressing inflammatory mechanisms.        Hypothermia may be effective because of this so-called “dirty”        neuroprotection as compared with drugs that block only one        aspect of the ischemic cascade

Treatment of Other Disease Conditions Using IAIPs

In addition to providing neuroprotection (in adults and neonates) andtreating tissue ischemia (e.g., in the brain), IAIPs can also beadministered to a subject (e.g., a human) for the treatment of burns.IAIPs can also be administered for the treatment of influenza (e.g.,H1N1 flu, bird flu, or other influenza strains known to cause disease inhumans). Other viral infections that can be treated by administering anIAIP include, e.g., Dengue fever and West Nile fever.

Animal Model of Disease in Humans

The ovine fetus has been widely used to investigate brain development(A59-A61). The neurodevelopment of the immature ovine brain is similarto that of the premature infant with respect to completion ofneurogenesis, onset of cerebral sulcation, and detection of the corticalcomponent of the auditory evoked potentials (A59, A62, A63). Full termin sheep pregnancy is 148 days of gestation. The preterm fetal sheepbrain between 94 and 96 days of gestation is comparable to that of thepreterm infant between 24 and 28 weeks of gestation, whereas fetal sheepat 135 days of gestation is similar to that of the near term humaninfant (A64). We examined sheep over a wide range of ages to have abroad developmental range, over which to examine changes in IAIPsexpression in brain and somatic organs. We examined fetal sheep at87-90, 105-108, and 135-137 days of gestation, which represents 60% or70%, 90% of the ovine gestation, newborn lambs, and adult sheep.Although rodents are frequently used to study brain development, therodent brain is immature at birth (A64) and almost completely agyric. Incontrast, similar to the non-human primate and human brain, the sheepbrain develops prenatally and is gyrencephalic.

In view of the similarities in the development of sheep and neonatebrains, sheep represent a useful model for examining the effectivenessof IAIP therapies in neonates and human subjects generally.

In the present invention, IAIP levels are measured and correlated tohealthy or injured brain post hypoxic ischemia. Previous work by Yow-PinLim, M.D., Ph.D. has clearly shown strong correlation between IAIPlevels and systemic inflammation, where systemic inflammation iselevated when IAIP levels are depressed. Or it could be said thatsystemic inflammation occurs because IAIP levels are depressed. Thepresent inventors recognized that ischemia/stroke represents anon-infectious cause of brain inflammation, and thus discovered thatbecause IAIP levels correlate to brain ischemia/stroke, patients at riskof, or experiencing, ischemia/stroke can be treated by administration ofan IAIP.

The following examples are provided by way of illustration only and notby way of limitation. Those of skill in the art will readily recognize avariety of non-critical parameters that could be changed or modified toyield essentially the same or similar results.

Examples Example 1: IAIPs Attenuate Ischemic Brain Injury in the OvineFetus

We exposed fetal sheep to ischemia/reperfusion injury.^(4, 5, 88)Sections stained with Luxol fast blue-hematoxylin/eosin (LFB-H&E) todelineate white matter lesions showed homogeneous blue stained myelinand healthy appearing cerebral cortex in control (FIG. 4A,1×) incontrast to ischemic (FIG. 4B,1×) brains that exhibited decreased bluestaining and cerebral cortical thinning indicating severe white matterand neuronal loss, respectively. Fetal sheep treated with IAIPs (4 mg/kg15 min, 24 & 48 h after carotid occlusion, FIG. 4C,1×) showed remarkablepreservation of white matter and cerebral cortex. A pathologist, unawareof treatments, scored the sections according to the percentage ofneuronal and white matter destruction using a grading system that wepreviously reported (FIG. 6).^(4, 5, 88) The pathological scoresindicated severe cerebral cortical and white matter injury in fetusesexposed to ischemia/reperfusion (closed bars) compared with control(open bars; P=0.07) and IAIP-treated ischemia/reperfusion (hatched bars,treatment as above). We also identified dramatic ischemia-relateddecreases in MBP and altered cellularity of GFAP positive astrocytes inthis model.⁵ Our findings suggest that treatment with IAIPs have greatpotential as a neuroprotective agent in the perinatal period andprobably also for other age groups.

Example 2: Ontogeny of Inter Alpha Inhibitor Proteins in Ovine Brain andSomatic Tissues IAIPs Detection by ELISA and Western Immunoblot inPlasma

IAIPs detected by the sheep specific ELISA in ovine plasma were lower(P<0.05) in the fetuses at 70% and 90% gestation than in the newbornlambs, and lower in the fetuses at 90% of gestation than in adult sheep.

The IAIPs were detected as 125 kDa and 250 kDa bands in ovine plasma byWestern immunoblot. The expression of 125 kDa band did not differ amongthe age groups. In contrast, the expression of 250 kDa band was lower inthe fetuses at 70% and 90% gestation, and in the newborn lambs than inthe adult sheep.

IAIPs detection by Western immunoblot in cerebral cortex, CP and CSF.IAIPs were detected in cerebral cortex, CP and CSF as 125 kDa and 250kDa protein bands by Western immunoblot using the specific antibodyagainst IAIPs. The expression of 125 kDa band was higher in the cerebralcortex in fetuses at 70% and 90% of gestation than in the newborn lambs,and higher in the adult sheep than in the newborn lambs. In contrast,the 250 kDa protein band expression in was lower in the cerebral cortexin the fetuses at 70% and 90% gestation and in the newborn lambs than inthe adult sheep. The expression of the 125 kDa band in CP was lower innewborn lambs than in the fetuses at 60% and 90% of gestation and in theadult sheep and the 250 kDa protein expression was higher in the fetusat 60% and 90% gestation and in the adult sheep than in the newbornlambs, but lower in the fetuses at 60% and 90% gestation than in theadult sheep. The expression of the 125 kDa and 250 kDa protein bands inCSF were higher in fetuses at 70% and 90% of gestation than in thenewborn lambs. The IAIPs levels in the CSF were below the limit ofdetection by the sheep specific ELISA. In summary, the cerebral cortex,CP and CSF each exhibit distinct patterns of expression for the 125 kDaand 250 kDa proteins. However, both molecules appear lower in thenewborn lambs than in the fetuses at 70% and 90% of gestation in both CPand CSF. We do not know the pattern of expression in adult sheep as wedid not have samples from the adult sheep.

IAIPs Detection by Western Immunoblot in Somatic Tissues.

The 125 kDa and 250 kDa IAIPs are expressed in different somatic organs.IAIPs were also detected as 125 kDa and 250 kDa proteins in placenta,liver, heart and kidney in fetal, newborn and adult sheep. In placenta,the 125 kDa band expression was lower at 70% than at 90% of gestation,but the 250 kDa expression did not differ between the fetuses at 70% and90% of gestation. In the liver, 125 kDa and 250 kDa band expressionswere lower in the fetuses at 70% and 90% gestation and in the newbornlambs compared with the adult sheep. In the heart, the expression of 125kDa band was higher in the fetuses at 70% gestation than in the fetusesat 90% of gestation, in the newborn lambs and in the adult sheep. Incontrast, the 250 kDa band did not differ among the groups. In thekidney, the 125 kDa band expression was higher in fetuses at 70%gestation and in the adult sheep compared with the fetuses at 90% ofgestation and the newborn lambs, but the expression of 250 kDa band waslower in fetuses at 70% and 90% of gestation and in the newborn lambsthan in the adult sheep.

DISCUSSION

The purpose of our study was to examine the expression of IAIPs in thebrain and in somatic organs of sheep during development as an initialapproach to understand these critical molecules during development. Thepresence of IAIPs was identified for the first time in plasma, cerebralcortex, CP, liver, heart, and kidney from early in fetal and through theneonatal period up to maturity in adult sheep, and in the placenta andCSF during fetal life as both the 125 kDa and 250 kDa proteins. Thefindings of our study are novel because to the best of our knowledgeprevious work has not reported distributions of IAIPs in the brain andsomatic organs during a wide span of development in any species. Themajor findings of this study were as follows. 1. The concentration ofIAIPs increase in plasma after birth. 2. The 125 kDa expression of IAIPswas higher in the adult and fetal than in newborn lamb cerebralcortices, but the 250 kDa protein expression was higher in adult thanfetal and newborn cerebral cortices. 3. The expression of IAIPs in CPwas highest in the adult sheep. 4. IAIPs were high in CSF of fetal sheepand very low in newborn lambs after birth. 5. IAIPs exhibit ontogenicpatterns of expression specific to each molecular species and organ. Thepresence of both molecules of IAIPs with organ specific patterns ofexpression during ovine development may be interpreted to suggest thatthese proteins have important immunomolatory (1, 2) functions duringorgan development.

Recent studies have shown high levels of circulating IAIPs are normallypresent in adult human plasma (2, 54) and even in plasma of prematureinfants (6-8). Our finding during ovine development extend theseobservations in human plasma and suggests the concentrations of IAIPs,measured by ELISA, increase markely after birth. In addition, theexpression of the IAIP-related molecules, which contribute to the totalamount of IAIPs measured by ELISA, differ with respect to theirexpression during development such that the expression of the 125 kDamoiety is similar at all ages, but that of the 250 kda protein increasesmarkedly after birth suggesting that it is the 250 kDa moiety thatcontributes to the high levels of total IAIPs observed in adult sheepplasma. However, although the level of the total IAIPs (ELISA) are highin the newborn lambs, the expression of the 250 kda protein appears lowin lambs, suggesting that the 250 kDa moiety cannot account for the highlevels of the total IAIP protein after birth.

IAIPs related proteins have previously been localized in various tissuesin adult rodents and humans, including cerebrum and cerebellum, lung,liver, intestines, colon, kidney, bladder, testes, and skin (22, 74-76).IAIPs also have been shown to have a specific distribution within thebrains of mice and rats with localization primarily in the cerebralcortex, hippocampus and hypothalamus (77). Unfortunately, we only hadresidual cerebral cortical and CP samples from our previous studies (61,65-68) so that we cannot comment on the amounts of IAIPs expressed inother brain regions. However, in the cerebral cortex, we observed adistinct ontogenic pattern for the 125 kDa and 250 kDa moieties, suchthat the former was higher in the fetuses and the later higher in theadult sheep. Although we cannot comment upon the distribution of IAIPsin other brain regions, identify the localization of IAIPs to specificcell types or identify the biological functions of IAIPs from our study,others have reported that IAIPs are most likely produced within theneurons (77) and/or astrocytes (29) in the murine brain, because intenseimmunoreactivities were localized to neuronal processes.

Inflammation plays a key role in many CNS disorders (78). There is nowevidence to suggest that bidirectional communications between the CNSand periphery could contribute to acute and chronic CNS disorders (78).Increased levels of IAIPs in ovine plasma and CNS tissue duringdevelopment could be related to the importance of these molecules insystemic and CNS inflammatory and immunological responses (1, 2). Recentevidence suggests that bikunin, the light chain of IAIPs, reducesoxidative stress, early inflammation, and endothelial activation in theforebrain of rats (79), reduces ischemia-reperfusion-related delayedneuronal apoptosis in gerbils (80), protects against white matterdemyelination and oligodendrocytes from apoptosis, and promotesremyelination in a model of experimental autoimmune encephalomyelitis(32). In addition, bikunin attenuates polymorphonuclear neutrophilinfiltration and decreases infarct volume in ischemic-reperfusion injuryin the brain of adult rats (31). Moreover, endogenous IAIPs appear to bedirectly involved in repair process of injured neurons (31) and proteaseinhibitors derived from neuronal cells function as regulators of neuriteregeneration and outgrowth (81). Hence, IAIPs appear to have a varietyof important neuroprotective effects in several animal models.Therefore, based upon our findings identifying the presence of IAIPs inrelatively large amounts throughout ovine development, we speculate thatthese molecules could potentially represent endogenous anti-inflammatorymolecules with neuroprotective properties.

The patterns of IAIP expression in the choroid plexus were somewhatsimilar to those of the cerebral cortex during development. CSF isproduced as an ultrafiltrate of plasma by the choroid plexus and alsofrom drainage of interstitial fluid from CNS tissues. Approximately 80%of the total amount of protein in CSF originates from blood with theremaining 20% originating directly from the CNS (82). CSF in adults hasmuch lower protein concentrations than plasma due to restricted entry ofblood derived components through the blood-CSF barrier (40). Most of thehighly abundant proteins in plasma are also elevated in CSF withexception of those proteins forming large complexes resulting in verylow diffusion rates into CSF(40). High concentrations of protein havebeen previously reported in the immature CSF of fetal sheep (57) and innewborn and preterm infants with levels several times higher than thoseof adults (83). The higher protein concentrations in fetal CSF are mostlikely a result local production by CP rather than immaturity of theblood-brain or blood-CSF barriers because the blood-brain and blood-CSFbarriers form very early during development in the fetus (40, 56,83-85).

The high levels of both the 125 kDa and 250 kDa IAIP protein moietiesexpressed in CSF in the fetal sheep, which decrease after birth, areconsistent with findings of elevated levels of other proteins duringgestation in several other species including rodents, pigs, rabbits,chickens and in premature infants (56, 83, 86-89). Although initially itwas thought that elevated protein concentrations in CSF simply reflectedan immature leaky blood-brain barrier to proteins during development,more recent information suggests that elevated CSF proteins in the fetusand newborn most likely have important roles for brain growth anddevelopment (40). The protein composition of CSF in the early stages offetal development is very complex. The majority of proteins are lowmolecular weight proteins such as albumin, alpha-fetoprotein,transferrin, lipoproteins etc., the concentrations of which showsignificant variations during different stages of development (56, 90).These protein fractions most likely represent molecules that haveimportant biological functions including growth factors and cytokines,which could influence the development of neuroepithelial cells (40, 86,91). The ontogenic patterns of protein concentrations in fetal CSF havebeen studied in several species. In the chick and sheep, proteinconcentrations increase consistently during the late fetal period anddecrease just before delivery (56, 92, 93). In contrast, this decreasedoes not occur until after birth in rats (55), suggesting thatphylogenic differences play a role in the pattern protein expression inCSF during maturation. Differences between patterns of proteinconcentrations in sheep and rodent CSF most likely result frommaturational differences in brain development among species (94). Alarge proportion of brain development in the sheep occurs before birthand, similar to the human, the sheep exhibits two distinct phases ofbrain growth (94, 95). The first phase occurs between 40 and 80 days ofgestation and is thought to represent neuronal multiplication and thesecond phase occurs between 95 and 130 days of gestation and representsneuralgia multiplication and myelination (62, 94, 95). In contrast, themajority of the rodent brain growth occurs after birth (Dobbing et al1979). These differences could also influence the expression of CSFproteins during development.

IAIPs have been previously detected in human CSF in patients with braintumors and inflammatory diseases, but their levels were not affected bylevels of systemic bikunin (96). CSF proteins may originate from severalsources including plasma, brain parenchyma and choroid plexus secretion.Both the in immature and adult subjects, CP synthesizes a large numberof neuropeptides, growth factors, and cytokines (91). Numerous studiessuggest that the high concentration of protein in fetal CSF is not dueto simple diffusion from plasma, rather there are specificdevelopmentally regulated transfer mechanisms in the CP (42, 97-103).Inspection suggests that this phenomenon is true in sheep as the CSFIAIP levels are higher in the fetuses than in newborn lambs, but theplasma IAIPs levels are higher in the newborn lambs than in fetuses.Therefore, we speculate that the presence relatively high levels ofIAIPs in fetal CSF is probably due to local synthesis by the CP andbrain tissue during critical periods of brain development, and thatthese molecules in CSF could be important in brain development in thefetus. Although we cannot discern from our study the reason the levelsof IAPs decreased dramatically after birth, we speculate that the stressof delivery along with endogenous hormonal changes could have affectedthe CSF levels of IAIPs after birth.

Endogenous IAIPs were detected for the first time during the developmentin the sheep CNS. They were detected in relatively high amounts in thecerebral cortex and CP at all stages of development and in the CSFduring fetal life. However, expression in cerebral cortex, CP and CSFdecreased in newborn lambs after delivery. We speculate that therelative reductions in IAIPs in the newborn lambs after birth couldrelate to the stress of delivery. The levels in cerebral cortex and CPincreased again in adult sheep, most likely related to the importance ofthese proteins in innate immunity. Although we cannot be certain of thephysiological significance our findings of high IAIPs levels in ovinebrain, CP, and CSF during the development, our findings raise theinteresting possibility that they are important molecules for braindevelopment.

Similar to our findings in the brain, we have shown for the first timethat these immunomodulatory proteins are present in somatic organs andin the placenta, and that they exhibit molecular weight and organspecific patterns of developmental regulation in liver, heart, kidneyand placenta. Although the exact functions of IAIPs are not known, theirpresence in large amounts with organ specific variations duringdevelopment raises the possibility that they represent endogenousanti-inflammatory molecules with organ specific differential productionor modulation during development.

There are several limitations to our study. We did not have CSF samplesfrom adult sheep available and, consequently can not compare adultvalues with those of the fetuses and newborn lambs. However, IAMPs arenot detectable in CSF from healthy adult humans (Y. P. Lim, personalcommunication, non-published data, 2012), but arc increased in thepresence of inflammation and tumors (96). We also did not have samplesproperly saved from our previous studies to determine the specificimmunohistochemical location of IAIPs the brain and we did not havetissue available from other brain regions. Consequently, we cannotcomment upon the cellular localization of IAIPs or on their expressionin other brain regions.

CONCLUSIONS

We conclude that IAIPs exhibit specific patterns of expression in theCNS and somatic organs of sheep during development. Although exactfunctions of IAIP are not known in CNS and somatic tissues, theirpresence in high amounts during development suggests their importance tobrain and organ development.

Materials and Methods

The present study was conducted after approval by the InstitutionalAnimal Care and Use Committees of Brown University and Women & InfantsHospital of Rhode Island and according to the national Institutes ofhealth Guidelines for use of experimental animals.

Animal Preparation and Experimental Design

Plasma, cerebral cortical, CP, CSF, placenta, liver, heart and kidneytissues samples for the present study were frozen samples obtained fromplacebo treated sham operated control animals from previous studies (61,65-67). Samples from all age groups were obtained over similar timeintervals. Surgical procedures and physiological measures were performedfor the former studies (61, 65-68). As described previously in detail(61, 65-67) surgery was performed under ketamine (10 mg/kg) and 1%-2%halothane anesthesia in pregnant ewes at 60% (87-90 days), 70% (106-107days), 90% (135-138 days) of gestation, newborn lambs (4-6 days of age)and adult non pregnant sheep (3 years of age). Plasma samples wereobtained from all animals just before the euthanasia. All animals weresham-operated control animals from our previous studies and sacrificedwithout further intervention. At the end of the studies, a CSF samplewas obtained from the fetal and newborn sheep with a direct puncture ofthe allantoic membrane. The sample was inspected for blood contaminationand discarded if there was evidence of contamination. CSF samples werenot available from the adult sheep. Tissues, plasma, and CSF were snapfrozen in liquid nitrogen and remained at −80° C. until analysis.Although choroid plexus samples were not available from fetuses at 70%gestation, samples were available from fetal sheep at 60% gestation.

Competitive ELISA to measure IAIPs level in ovine plasma and CSF IAIPsconcentrations were measured by specially developed competitive ELISA insheep plasma using a polyclonal antibody against human IAIPs (R-16 pAb).The polyclonal antibody was generated by immunizing rabbits with highlypurified human plasma derived IAIPs. The R-16 pAb cross-reacts withnon-human IAIPs including sheep. 96-well high binding microplate platesMicrolon 600 (Greiner Bio-One, Monroe, N.C., USA) were coated withpurified sheep IAIPs. Sheep IAIPs were purified from sheep serum (QuadFive, Ryegate, Mo., USA) by anion-exchange chromatography on a ToyopearlQ-600C-AR column (Tosoh Bioscience, King of Prussia, Pa., USA). BoundIAIPs were eluted with a buffer containing 750 mM NaCl. The purifiedsheep IAIPs were diluted in 100 mM NaP04 buffer pH 6.5 and immobilizedon the microplates (50 ng/per well) for 1 h at room temperature orovernight at 4° C. Subsequently, the microplate was blocked with 200 μLof 5% non-fat dried milk in PBS and 0.05% Tween. Sheep plasma wasdiluted in PBS and a known amount of purified sheep IAIPs was seriallydiluted in PBS containing 1% BSA to establish a standard curve forquantitative analysis of IAIP concentrations in the samples. After 50 μLof samples and serially diluted IAIPs standards were added to the wells,50 μL of R-16 pAb diluted in 1:1200 in PBS was added to each well.Plates were incubated for 1 h at room temperature and subsequentlywashed with PBS and 0.05% Tween using automated plate washer (BiotekEL-404, Winooski, Vt., USA). The bound R-16 pAb was detected by addingHRP-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, Calif., USA)for 1 h at room temperature. After washing, 100-μL Enhanced K-Blue TMBsubstrate (Neogen Corp, Lexington, Ky., USA) was added to the wells andthe reaction was stopped by adding 100 μL 1N HCl solution. Theabsorbance at 450 nm was measured on SpectraMAX Plus microplate reader(Molecular Devices, Sunnyvale, Calif., USA). Each sample was tested intriplicate and assays were repeated at least twice on all samples.

Preparation of Cytosolic Tissue Fractions

Cell cytosolic fractions of cerebral cortex, CP, placenta, liver, heart,kidney for IAIPs were extracted in buffer A (TRIS 10 mM pH 6.8, Sucrose,MgCl) with one percent complete protease inhibitor cocktail (Sigma, St.Louis, Mo., USA). Total protein concentrations of the homogenates weredetermined with a bicinchoninic acid protein assay (BCA, Pierce,Rockford, Ill., USA). Aliquots of the extracted samples were stored at−80° C.

Western Immunoblot Detection and Quantification of Proteins

Fifteen to fifty μg protein of total protein per well (cerebral cortex:50 g, choroid plexus: 15 μg, cerebral spinal fluid: 22.5 μl, plasma: 1μl from 1:100 dilution; placenta: 30 μg, liver: 50 μg, heart: 50 μg andkidney: 50 μg) were fractionated by SDS-PAGE electrophoresis andtransferred onto PVDF membranes (0.2 micron, Bio-Rad Laboratories,Hercules, Calif.) using a semi-dry technique. Membranes were incubatedwith IAIP primary rabbit polyclonal antibody (ProThera Biologics, EastProvidence R.I., USA) at a dilution of 1:5,000. The immunoblots wereincubated in primary antibody overnight at 4° C. Peroxidase-labeledsecondary antibody goat anti-rabbit (Alpha Diagnostic, San Antonio,Tex., USA) was incubated for 1 h at room temperature in a dilution of1:10,000. Binding of the secondary antibody was detected with enhancedchemiluminescence (ECL plus, Western Blotting Detection reagents,Amersham Pharmacia Biotech, Inc., Piscataway, N.J., USA) before exposureto autoradiography film (Daigger, Vernon Hills, Ill., USA).

Experimental samples were normalized to a reference protein standardthat was obtained from a homogenate protein pool from the tissues of asingle adult sheep. For the purpose of this report, we refer to thesesamples as internal control samples. As we have previously described(69-72), these samples served as an internal control for quality ofloading, transfer of the samples, normalization of the densitometricvalues, and to permit accurate comparisons among the differentimmunoblots (69, 70, 73). The use of internal control is unique to ourlaboratory and allows us to compare large groups of animals over a largenumber of different immunoblots. We developed this methodology becauseinvestigation of a large number of housekeeping proteins showed thatthey all exhibited significant variations during ovine developmentmitigating their use as house keeping proteins. The experimental proteinautoradiographic densitometrical values were expressed as a ratio to theinternal control, thus facilitating normalized comparisons amongdifferent groups and immunoblots. When this methodology was used withina single age group (newborn), the method correlates well with valuesthat were normalized as ratios to β-actin (69).

Each immunoblot included samples from the four groups and three internalcontrol samples. The internal control samples were included in threelanes, as the first, middle, and last samples on each immunoblot. Wecalculated a coefficient of variation for the internal control sampleson each immunoblot. The values for the experimental samples wereaccepted as valid only if the percent coefficient of variation for theinternal control samples was less than 20% on the immunoblot. Humaninter-alpha-inhibitor protein served as a positive control for allimmunoblots to ascertain that the antibody correctly identified theovine proteins. Molecular weight standards (Bio-Rad Laboratories,Hercules, Calif. USA) were included in each immunoblot. The primaryrabbit polyclonal anti-IAIP detected IAIPs bands at 125 and 250 kDa inall organs. Uniformity in inter-lane loading was also established byCoomassie blue (Sigma, St. Louis, Mo., USA) staining of thepolyacrylamide gels and uniformity of transfer to the polyvinylidenedifluoride membranes was confirmed by Ponceau S staining (Sigma, St.Louis, Mo., USA). For the purpose of illustration in the figures, weselected the immunoblot that most closely represented the mean valuesfor each age group and tissue from the different immunoblots.

Densitometric Analysis

Band intensities were analyzed with a Gel-Pro Analyzer (MediaCybernetics, Silver Spring, Md., USA). All experimental samples werenormalized to the respective average of the three internal controlsamples. However, the band intensities were expressed as arbitraryoptical density units for CP and CSF as we did not have adult CP or CSF.The final values represented averages of the densitometry valuesobtained from the different immunoblots (plasma n=2; cerebral cortexn=8; choroid plexus n=5; cerebral spinal fluid n=2; placenta n=5, livern=5; heart n=5; kidney n=5) and were presented as a ratio to theinternal control sample except for CP and CSF.

Statistical Analysis

All results were expressed as means±SEM. Two-way analysis of variance(ANOVA) was used to compare the differences among the groups. Thefactors were age group (fetuses at 60%/70%, 90% of gestation, newborn,and adult) and protein expression (125 kDa and 250 kda band). Whensignificant difference was detected by ANOVA, the Fischer leastsignificant difference test was used to further describe thestatistically significant differences among the groups. P<0.05 wasconsidered statistically significant.

Example 3: IAIPs Provide Neuroprotection Prior to Stroke and FollowingHypoxia/Ischemia in Neonates

Neurological impairment secondary to oxygen deprivation, includinghypoxia/ischemia (HI) associated with immaturity of vasculature andpulmonary insufficiency in premature and very low birth-weight infants,as well as HI events relating to birth, is the leading cause ofneurologic morbidity and mortality in infants. Affected children areprone to long-term cognitive and behavioral deficits. Moreover, severityof injury and pathological outcome are dependent upon sex, with moresubstantial long-term deficits identified in male than female infants,even when matched for severity of injury. The cause(s) of thesedifferences are largely unknown; however, data indicate sex differencesin apoptotic mechanisms, suggesting sex-specific mechanism of HI-inducedinjury. We propose novel studies to examine this sex-based phenomenon,specifically with regard to underlying molecular/cellular features of anHI event. We will examine the role of pro-inflammatory cytokines (knownto cause/accentuate brain injury) and inter-alpha inhibitor protein(IAIP, known to effectively down-regulate cytokines) in histological andlong-term behavioral studies of male and female rats with HI. Excitingpreliminary data suggest substantial decreases in IAIP acutely followingbrain ischemia in fetal sheep, as well as significant neuroprotection byIAIP in male HI rats. These findings, combined with previous datasuggesting neuroprotection from pre-stroke IAIP treatment in adult rats,raise the possibility that exogenous IAIP following neonatal HI mayrepresent an effective therapeutic strategy. However, given evidence ofsex differences in long-term outcome following neonatal HI, it remainsto be seen if male and female brain varies in cytokine activation andIAIP expression following injury, whether IAIP will proveneuroprotective to both sexes, and the mechanisms of suchneuroprotection. Through histological and long-term behavioral study ofboth male and female rats, the proposed studies determine sexdifferences in cytokine and IAIP expression after HI and the modulationof key sex differences in cell death mechanisms by IAIP. Additionally,IAIP is poised to enter clinical trials for sepsis and related moleculesare known to suppress preterm labor, thus, this therapeutic agent couldrapidly enter clinical use to attenuate HI injury in infants, though wepredict a sexually dimorphic response due to key modulators involved insex-specific mechanisms of HI-related cell death.

Example 4: IAIP Administration

Systemic administration of IAIPs, or their cleavage products, reducesthe production of pro-inflammatory cytokines and prevents/attenuates thedevelopment of ischemic-reperfusion injury in the immature brain. Usinga highly reproducible model of brain ischemia-reperfusion in the ovinefetus, we have determined that treatment of the sheep fetus with IAIPsprevents/attenuates ischemia-related damage to the immature brain.Findings from our studies translates into an important novel treatmentstrategy for human infants with brain ischemia, as IAIPs can be preparedfrom human plasma, are in the development to treat adults with shocksyndrome/sepsis, and similar agents are efficacious in inhibitingpreterm delivery through suppression of cytokines and inflammatorymediators.^(8-10, 12-16) Consequently, it will be feasible to use IAIPsas anti-inflammatory immunomodulators similar to the manner in whichimmunoglobulins and fresh frozen plasma are currently used in infants.

We have examined the neuroprotective effects of systemic IAIPadministration on brain ischemia in the ovine fetus. Our data suggestthat IAIPs have important neuroprotective effects on ischemia-relatedcerebral cortical and white matter damage in the fetus (FIG. 7).

Example 5: Use of IAIPs in Treatment of Brain Damage in Premature Infant

There is an increasing incidence of premature birth, which contributesto 50% of cases of mental retardation and CP2. The incidence of CP2 is40-148/1,000 in premature and 1-2/1,000 in full-term infants.¹⁷⁻²⁰Although many infants who develop CP2 may be asymptomatic atbirth,^(21, 22) substantial evidence suggests antecedents of CP2 beginduring fetal life.²³⁻²⁶ Findings suggest that elevated cytokines areimportant in the pathogenesis of CP2.^(22, 27-30) Periventricularleukomalacia (PVL) is a white matter lesion in premature neonates thatis predictive of CP2.^(31, 32) Although the etiology of this disorder ismultifactorial, hypoxia-ischemia (HI) and overproduction ofpro-inflammatory cytokines represent underlying factors.^(×)Moreover,inflammatory processes that begin in utero are likely antecedents ofbrain damage in premature infants because early elevations ininflammation-related proteins, including cytokines, predict the risk ofsonographic white matter damage.⁴ However, this area remainscontroversial, as some have suggested neonatal infection and hypotensionare more significant risk factors for white matter damage thanchorioamnionitis.² Nonetheless, cytokines likely represent a finalcommon pathway, activated by a variety of insults, which contribute toand/or exacerbate brain damage.³⁵ Systemic treatment with IAIPsrepresents a novel neuroprotective strategy that can down-regulate bothsystemic and central nervous system (CNS) cytokines to attenuate/preventwhite matter damage and CP2.

Cytokines Effects on the Brain

Although the brain was previously considered an “immune privileged site”not under the influence of the immune system. important links betweenthe brain and immune system are now recognized^(36, 37) Cytokines areexpressed at low levels in normal brain, but CNS injury increasesvascular and parenchymal expression.³⁶ IL-1, TNF-α, INF-α, IL-6, INF-γand IL-8 are important in CNS inflammation,³⁸ which results inliquefaction and/or glial scars, as neurons do not proliferate.³⁸Pro-inflammatory cytokines, including IL-1, IL-6, and TNF-α, in brainparenchyma promote changes that accentuate brain injury.³⁹⁻⁴⁴

Pathogenesis of CP2 & PVL: Role of Cytokines in Ischemia-Reperfusion(I/R) Injury

There are two main theories of pathogenesis of PVL/CP2. In the classictheory, HI results in damage to white matter,⁴⁵ but an alternativehypothesis places cytokines central to mechanisms of brain damage.⁴⁶Intravascular cytokines are elevated in full-term infants who developCP2,²² amniotic fluid cytokines and cord blood IL-6 are increased inpremature infants who develop white matter lesions,^(28, 30) andpro-inflammatory cytokines are detected in white matter lesions ofinfants who died with PVL.²⁹ Evidence also suggests systemicinflammation, sepsis, and necrotizing cnterocolitis (NEC) are associatedwith increased incidences of CP2, lower mental and psychomotordevelopment, and visual impairment.^(47, 48) Both NEC and sepsisincrease the risk for inflammatory-mediated white matter damage.⁴⁷⁻⁴⁹IAIPs attenuate ischemia-related white matter damage, as suggested byour data;⁵ IAIPs act by reducing ischemia-related increases in cytokinesin the brain.⁵⁰

Inter-Alpha Inhibitor Proteins, Systemic Inflammation & Tissue I/RInjury

IAIPs are a family of structurally related proteins found in plasma inhigh concentrations. IAIPs are important in inflammation, wound healingand cancer metastasis.^(51, 52) The major forms in human plasma areInter-alpha inhibitor (IaI), which consists of two heavy chains (H1 &H2) and a single light chain, and Pre-alpha Inhibitor (PaI), consistingof one heavy (H3) and one light chain. The light chain (bikunin)inhibits several serine proteases.⁵³ Liver is the major site ofsynthesis of heavy and light chains of IAIP.⁵⁴ High levels of IAIPsnormally in plasma of adults and newborns, even when born prematurely,indicate these proteins are essential.⁵⁵ No person with complete absenceof IAIP has ever been detected.⁵² Markedly decreased plasma levels inseptic patients and concomitant increases in IAIP-related fragments inthe urine suggest these proteins are ‘consumed’ and rapidly cleared fromsystemic circulation during sepsis. Hepatic IAIP synthesis is alsodown-regulated during severe inflammation. Although the physiologicalfunction of IAIPs remain to be established, current data suggests thesemolecules are part of the innate immunity and play a critical roleduring inflammation.⁵⁶

In addition to its broad anti-protease activity, IAIPs have uniqueimmunomodulatory effects in reducing TNF-α during systemicinflammation⁵⁷ and augmenting anti-inflammatory IL-10 in a neonatalsepsis model.¹¹ The light chain of IAIPs (urinary trypsin inhibitor(UTI), or bikunin), also effectively inhibits premature delivery thoughcytokine suppression and inflammatory mediators.^(8-10, 12-16) Althoughthe mechanism(s) by which IAIPs mediate biological functions remains tobe determined, recent discovery of pro-inflammatory stimulatedglycoproteins and TNF-stimulated gene 6 (TSG-6) suggests upon forming astable complex with TSG-6, one of the possible ligands of IAIP,inhibitory activity of IAIP toward plasmin is enhanced.⁵⁸

Plasmin is a serine protease that activates metalloproteinases (MMPs),which are a part of the inflammation-related proteolytic cascade. MMPsare important in neuronal cell death resulting from intracerebralhemorrhage, neuroinflammation-related neurotoxicity, andneurodegenerative disorders.^(59, 60) MMPs increase permeability of theblood-brain barrier resulting in edema, hemorrhage, and cell death.⁵⁹Therefore, the ability of IAIPs to inhibit plasmin activity and in turnreduce the activation of injury-related MMPs may represent one of themechanisms by which IAIPs could be neuroprotective.⁶¹

IAIPs and related molecules have been detected in neurons, astrocytes,and meningeal cells of the brain and, based upon their role in otherorgans, may function as endogenous neuroprotective molecules. Moreover,we detected IAIPs in the cerebral cortex (FIG. 4) of sheep duringdevelopment and in cerebral spinal fluid of ovine fetuses (CSF). Bikuninhas been shown to block TNF-α's production during the reperfusion phaseof ischemic injury in several organs (liver, kidney, heart, intestineand lung),⁶²⁻⁶⁴ however, there is very little information on thesemolecules in brain. We have recently shown decreases in IAIPs associatedwith ischemia-reperfusion in the ovine fetal brain (FIG. 3) and othersreport UTI attenuates stroke-related brain injury and experimentalautoimmune encephalomyelitis (EAE)-related white matter loss in adultrats.^(61, 65)

In premature infants, IAIPs decrease during sepsis⁶⁶ and NEC.⁶⁷ Inaddition, both disorders are associated with an increased incidence ofbrain damage, suggesting the interesting possibility that decreases inIAIPs levels contribute to the development of associated braindamage.^(47, 48) IAIPs attenuate ischemia-related white matter injury,as suggested by our data (FIGS. 4-6). IAIPs prevent inflammation-relatedwhite matter damage in the premature brain.

The subunit bikunin, purified from urine, has a very short half-life (3to 10 min) in the circulation. In contrast, IAIPs isolated from bloodrepresent native complexed forms of the protein, have a longer half-life(8-12 h), and thus, are more feasible as therapeutic agents. Theneuroprotective properties of this natural form have only been examinedin our studies and are likely to have considerably greater therapeuticefficacy than that of the bikunin subunit. ^(61, 65)

Sheep Model of Human Disease

The large amount of data on the fetal sheep brain is highly relevant toconditions in premature infants.^(1, 68-76) A review⁶ on the use ofinstrumented fetal sheep to define pathogenesis of human white matterinjury supports our original contention that the immature sheep fetus isan excellent model for study of brain maturation.⁷⁴⁻⁸⁰ The immatureovine brain (0.65 gestation or 95 days)⁶ is similar to that of thepremature human between ˜24 and 28 weeks with respect to neurogenesis,cerebral sulcation, and detection of the cortical component of auditoryand somatosensory evoked potentials.⁸¹⁻⁸⁴ Similar to findings inpremature infants, the immature ovine brain has limited capacity forcerebral autoregulation, immature white matter, and very high watercontent.^(6, 7, 85, 86) We, and others, have reported that white matterlesions similar to those in premature infants are more reproducible insheep fetuses than in rodents.^(5-7, 68) Importantly, the only majorprogress in prevention/attenuation of HI injury in human newborns was adirect result of studies done in the sheep fetus.^(1, 72)

Example 6: Neuroprotective Effects of Systemic IAIP Administration onBrain Ischemia in the Ovine Fetus

Our data demonstrate several important new results that suggest thefeasibility and probable successful outcome of our aims.

1. Purification of IAIP from Sheep Serum.

IAIPs will be extracted from ovine serum (Quad-Five, Ryegate, Mo.) usinganion-exchange chromatographic separations on Tosoh Q and monolithicDEAE-CIM columns (Tosoh Q-600C-AR, Tosoh Bioscience, King of Prussia,Pa. and DEAE Convective Interactive Media, BIASeparation, Austria). Wedeveloped efficient separation methods for a high yield and purity ofovine IAIPs. Approximately 25-liter ovine serum will be extractedyielding ˜4-5 g highly purified biologically active IAIPs for in-vivofetal sheep studies. The SDS-PAGE shows 125 & 250 kDa bands in purifiedIAIPs from sheep serum (FIGS. 8 and 9A/9B). Serum was passed toanion-exchanger columns and washed with salt and low pH buffers (Fr. 1and Fr. 2 of FIG. 8) before eluted in high salt (Fr. 3 of FIG. 8). SheepIAIPs in this eluted fraction are ˜85-90% pure. Western blot (WB)analysis with rabbit polyclonal antibody against IAIPs (R16) confirmedthe reactivity of IAIPs (125 kDa Pre-alpha Inhibitor & 250 kDaInter-alpha Inhibitor, arrows, FIGS. 8 and 9A/9B).

2. Detection of IAIP in Sheep.

To measure endogenous IAIPs quantitatively in biological fluids, weestablished a competitive ELISA using rabbit polyclonal antibodiesagainst human IAIPs that cross-react with ovine species. Purified ovineIAIPs are used to coat micro plates for ELISAs. Using known standardIAIP amounts, we established a linear standard curve. This ovine IAIPELISA will be useful in measuring IAIP levels in the studies below. Wehave shown that near term fetal sheep (90% gestation, 135 d, 55±27μg/ml, mean±SD) have lower (P<0.05) plasma IAIP concentrations thannewborn (111±38) and adult (102±46) sheep. IAIPs were also detected forthe first time in brain, CSF, and CP from early in fetal and throughoutovine development as both 250 kDa and 125 kDa proteins. Expression ofboth proteins were higher in adult than fetal brain (FIGS. 9A/9B,*P<0.05vs. adult, full-term gestation=148 d). In addition, high levels of IAIPsin fetal CSF and significant reductions (P<0.05) after birth suggesttheir importance to brain development. Although the functions of IAIPsin brain, choroid plexus, and CSF are not known, their presence in highamounts during development raises the interesting possibility that theyare endogenous anti-inflammatory-neuroprotective molecules and suggeststheir importance in brain development.

3. IAIPs Attenuate Ischemic Brain Injury in the Ovine Fetus and HI BrainDamage in Neonatal Rats.

We exposed fetal sheep to ischemia/reperfusion.^(4, 5, 88) Sectionsstained with Luxol fast blue-hematoxylin/eosin (LFB-H&E) to delineatewhite matter lesions showed homogeneous blue stained myelin and healthyappearing cerebral cortex in control (FIG. 4A,1×) in contrast toischemic (FIG. 4B,1×) brains that exhibited decreased blue staining andcerebral cortical thinning indicating severe white matter and neuronalloss, respectively. Fetal sheep treated with IAIPs (4 mg/kg 15 min, 24 &48 h after carotid occlusion; see, e.g., FIG. 4C, 1×) showed remarkablepreservation of white matter and cerebral cortex. A pathologist, unawareof treatments, scored the sections according to the percentage ofneuronal and white matter destruction using a grading system that wepreviously reported (FIG. 6).^(4, 5, 88) The pathological scoresindicated severe cerebral cortical and white matter injury in fetusesexposed to ischemia/reperfusion (closed bars) compared with control(open bars; P=0.07) and IAIP-treated ischemia/reperfusion (hatched bars,treatment as above). We also identified dramatic ischemia-relateddecreases in MBP and altered cellularity of GFAP positive astrocytes inthis model.⁵

Example 7: Confirm the Neuroprotective Effects of IAIPs onIschemic-Reperfusion Brain Injury in Fetal Sheep

HI increases systemic and local pro-inflammatory cytokines, which inturn potentiate HI brain damage in the perinatal period.^(91, 92) Instroke patients, elevated CSF cytokines⁹³ are associated with whitematter damage.⁹³ IL-1β, IL-6, and TNF-α mRNA have been detected aftercerebral ischemia in adult rats⁹⁴ and elevated cytokines were reportedafter HI in young rats.^(95, 96) Intracerebral IL-1β or TNF-α injectionsresult in brain injury in young rats, and IL-1β injures white matter.⁹⁷Intracerebroventricular injections of an IL-1 receptor antagonist reducecell death and caspase-3 activity in young rats after HI.⁹⁸ Thus,pro-inflammatory cytokines are upregulated by HI and damage the immaturebrain, and therefore reducing their activity attenuates injury-relateddamage. Further, IAIP treatment of newborn mice attenuatessepsis/inflammatory-related increases in systemic cytokines.¹¹ IAIPlight chain subunit or UTI is cytoprotective against liver, intestine,kidney, heart, and lung ischemic-reperfusion injury through itsanti-inflammatory activity, attenuates cerebral ischemia in an adultstroke model and white matter loss in an EAE model.^(61, 65) Althoughthese findings are encouraging, UTI has very short half-life (3 to 10min) in contrast to the native complexed IAIPs (8-12 h), thus makingIAIPs more useful as effective therapeutic agents. In the experimentsbelow, we show that IAIPs systemically administered after exposure toin-utero brain ischemia attenuate development of ischemia-related injuryin fetal brain. One of the mechanism(s) of potential neuroprotectiveeffects could be down regulation of pro-inflammatory cytokines in brainparenchyma.

TABLE 1 Gest. Age Placebo + Sham Placebo + Isch IAIP + Isch Reper. BrainTissue Subjects (days) (# Animals) (# Animals) (# Animals) (h) Exp. EndPoint Early Gest. 100-107 8 8 8 72 Pathology/Immunohist Late Gest.125-127 6 6 6 72 Biochem/Molecular Bio

Experimental Protocol:

Table 1 shows study subjects, gestational age at study, studyconditions, # of animals, duration of reperfusion, and tissue end-pointsfor the studies. Groups of early or late gestation fetal sheep will bestudied because both premature and near term human infants develop CP.Control fetal sheep will be exposed to placebo-sham ischemia,experimental to in utero brain ischemia (carotid occlusion: 30 min) withand without IAIP treatment during 72 h-reperfusion). Surgicalpreparation and carotid occlusion will be performed as we previouslydescribed.^(4, 5, 88) Brain tissue will be obtained for pathologicalassessment and scoring.

As shown in the schema of FIG. 13, after baseline measurements & 30 minischemia, fetal sheep will receive placebo or IAIP (4 mg/kg fetalweight) 15 min, 24 h and 48 h after the onset of reperfusion. Thistreatment regimen was selected because the 8-12 h half-life of IAIPsshould provide fetal exposure to IAIPs for the majority of the 72 hreperfusion, and this dose appears efficacious in our preliminary data.Measurements obtained at baseline, during ischemia, after 30 min ofischemia, and sequentially during reperfusion (FIG. 13, solid circles)include fetal heart rate, mean arterial blood pressure, amniotic fluidpressures, continuous ECoG, and separate sets of blood collection. Atthe end of the study, the ewe and fetus will be given intravenouspentobarbital (15-20 mg/kg) to achieve a surgical plane of anesthesiaand 200 mg/Kg of pentobarbital for euthanasia. Blood samples (FIG. 13,solid circles) are obtained for hematocrit, blood gases, oxygensaturation, arterial glucose and lactate, IAIP concentration, andcytokines (IL1-β and IL-6).⁹⁹

CSF samples are obtained for Western blot (IL1-β, IL-6, GFAP, MBP, andIAIPs). Coronal brain sections obtained at the level of the hypothalamus(mamillary bodies) for routine pathology (LFB-H&E) will be scored by apathologist unaware of treatment (E.G.S.) as we previouslydescribed.^(4, 5, 88) The remainder of the brain tissue will be used todetermine some of the mechanism(s) of action of IAIPs according to themethods described below. After this section is obtained, half of theremaining brain will be taken for frozen tissue and the contralateralhalf for immunohistochemistry (NeuN, IL1-β, IL-6, TNF-α, activatedcaspase-3 (see FIG. 10), MBP, and GFAP). Separate frozen sections ofcortex, caudate nucleus, cerebellum, hippocampus, thalamus, midbrain,and periventricular white matter will be obtained for Western blot (MBP,IL-1β, IL-6, TNF-α, and IAIP, MMPs) and ELISA (IL-6, IL-1β, caspase-3and IAIP).

Data Analysis.

Using group means from our preliminary data, assuming equal samplesizes, 8/group (n=24) would give 95% power at an alpha of p=0.05 todetect significant differences. Hence, we require 6/group for the late(to supplement our data) and 8/group for the early gestation sheep.Serial measurements will be compared by ANOVA for repeated measures withtime, treatment, and group as factors, brain samples for cytokines andMBP etc. by one-way ANOVA. If a significant difference is detected byANOVA, the Fischer LSD test will be used as a post hoc test. Apoptoticcells/mm² will be detected as described.¹⁰⁰ The # of apoptotic cells/mm²or NeuN positive cells will be analyzed by ANOVA andpathological/immunohistochemical samples by non-parametric methods.

Results-Interpretation.

We anticipate that IAIPs will reduce ischemic brain damage in the earlyand late gestation fetal sheep by at least 50% given our data. Weanticipate that there will be less seizure activity in the fetusesexposed to IAIP-brain ischemia compared with those exposed toplacebo-ischemia. We anticipate that ischemia-related white matter andneuronal injury will be attenuated in IAIP treated fetuses as suggestedin FIGS. 4-6.

Alternative Procedures.

Methodologies described above are routine in our labs. Although weelected to use the carotid occlusion model, an alternative model ofumbilical cord occlusion may be used in future studies to examine IAIPsbeneficial effects on brain and injury to other organs.³ The dosingprotocol that we selected appears beneficial. Other treatment doses,regimen, and durations may also be used.

IAIPs are large molecules that might not easily cross the blood-brainbarrier (BBB). However, IAIP-related molecules have been shown to beneuroprotective against focal cerebral ischemia-reperfusion injury⁶¹ andit remains possible that under pathological conditions, similar to thesituation with antibodies,¹⁰¹⁻¹⁰⁶ these molecules could enter the brainand have therapeutic effects. However, the two most likely mechanisms bywhich IAIPs could protect the brain are by reducing the concentrationsof intravascular cytokines generated during brainischemia/reperfusion,^(107, 108) and/or reducing ischemia-relatedincreases in endothelial derived cytokines that could leak into brainvia a damaged BBB to accentuate brain injury.¹⁰⁷ Nonetheless, werecognize that it would be of great interest to measure BBB permeabilitywith IAIPs under normal and pathological conditions.

Example 8: Examination of Some of the Mechanism(s) of Action andBiological Effects by which IAIPs Attenuate Ischemic-Reperfusion Injuryin the Immature Brain

Our data show that IAIPs exhibit mechanism(s) of action as aneuroprotectant in fetal brain. The approaches described herein can beused to further identify potential mechanisms by which IAIPs attenuatebrain damage in the fetus.

1. IAIPs & Ischemia.

We have shown that ischemia-reperfusion results in acute decreases inIAIPs 4 h after ischemia (I/R, *P<0.05 vs. control, FIG. 3), whichreturn toward control values (open bars) 24 & 48 h after ischemia. We donot know the mechanism for the reductions in IAIPs, but IAIP levels canbe measured in fetuses exposed to ischemia with and withoutIAIP-treatment to determine if IAIP brain expression is higher afterIAIP-treatment.

2. Ischemia Increases Pro-Inflammatory Cytokines in Ovine Fetus.

We recently reported using the same fetal model that cerebral corticalIL-1β was higher 48 & 72 h after ischemia compared with non-ischemicfetuses, and IL-1β & IL-6 levels were higher in white matter than incerebral cortex 72 h after ischemia.⁵⁰ Here we will examine theexpression of IL-1β, IL-6 & TNF-α in fetuses exposed to ischemia withand without IAIP treatment.⁵⁰

3. Ischemia Increases Caspase-3 in Ovine Cerebral Cortex.

Increases (*P<0.05 vs. Control) in caspase-3 were detected^(109, 110) 4,24 & 48 h after ischemia (FIG. 10). We do not know if caspase-3 iselevated 72 h after ischemia, but will measure it in the currentstudies.

4. Double-Label Immunofluorescence: Neuronal & Apoptotic CellularCounts. Caspase-3 is a key executioner of apoptosis.¹¹¹ We used NeuN isa neuronal marker,¹¹² measured DNA fragmentation (apoptosis) withterminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL), and all nuclei with DAPI in fetal brain to quantify neuronaland non-neuronal apoptosis in the ovine fetus.¹⁰⁰ FIG. 11 shows DAPIlabeled nuclei, NeuN positive nuclei (thick arrow), and a TUNEL positiveapoptotic nucleus (thin arrow). Merged lower portion shows a NeuNpositive nucleus, which is not apoptotic (thick arrow), and a nucleusexhibiting co-localization of green fluorescein apoptotic, red NeuNantigen, and blue DAPI nuclear DNA markers indicating this is anapoptotic neuronal nucleus (thin arrow).¹⁰⁰ Using these techniques, wewill quantify total number of surviving neurons and amount of apoptosisin neuronal and non-neuronal nuclei of ischemic brain tissue fromfetuses with and without IAIP treatment.¹⁰⁰

Our experimental approach will analyze some of the mechanism(s) by whichIAIPs exert their beneficial effects on ischemic-reperfusion braininjury in fetal sheep. Analyses below will be performed on the braintissue from fetuses. The purpose is to begin to elucidate some of thebiological effects of IAIPs and potential mechanism(s) underlying theneuroprotection suggested by our data. To this end, we will usemethodologies we published or are available in ourlaboratories.^(5, 100) This approach addresses three key questions basedupon our data: 1) Does exogenous IAIP administration affect brain tissueIAIP & CSF levels? 2) Do IAIPs quantitatively attenuateischemia-reperfusion-related neuronal and white matter loss? and 3) DoIAIPs attenuate ischemia-reperfusion related increases in cytokines andMMPs?

TABLE 2 Brain Regions Hippocampus, Caudate-Putamen, Frontal & ParietalCortex White Matter Regions (WM) Morphological- Subcortical & Deep WM,Corpus Callosum Immunohistochemical Biochemical/Molecular MorphologicalBiochemical/molecular Neuronal cell counts DNA fragmentation, MBP, GFAP,PLP DNA fragmentation, (NeuN), In situ DNA Caspase-3 activity, Caspase-3activity, fragmentation (ApopTag), MMPs 2, 3 & 9 Western blot: IAIPsCaspase-3 Western blot: IAIPs, Caspases, MBP, PLP, Casp. 3, MBP, GFAP,GFAP, IL-6, IL1β, IL-6, IL1β, TNF α, IL-6 TNF α

We will also determine some of biological effects/mechanisms of IAIPsprotection in brain ischemia. We will examine specific brain and whitematter regions as listed for neuronal and white matter (WM) markers ofinjury in tissue from fetal sheep exposed to sham control or ischemiawith and without IAIP treatment. We will examine brain (hippocampus,caudate-putamen, frontal, and parietal cortex, Table 2) and white matterregions (subcortical and deep WM, corpus callosum) that could beinfluenced by IAIP treatment.^(4, 5, 88) Western blot will be used tomeasure IAIPs in brain (FIGS. 3, 8, and 9A/9B) and CSF, cytokines as wedescribed,⁵⁰ proteolipid protein (PLP) as described,¹¹⁵ MBP and GFAP,after myelin isolation, with standard techniques.¹¹⁴

Data Analysis:

Multivariate ANOVA will be used to assess differences between totalnumber of dying cells, total # of neurons, apoptotic neurons, and othermarkers in the Table 2 across brain regions among experimentalconditions (control, placebo-ischemia and IAIP-ischemia), and one-wayANOVAs for individual brain regions for Western blots, MMPs, caspaseactivity etc. Post hoc testing will be similar to Aim1. These analyseswill reveal any subtle differences in brain markers in the Table 2across treatment conditions.

Results-Interpretation.

Based upon our data, we expect that neuronal loss will be less, totalneuronal number (NeuN) higher, apoptosis/caspase-3 lower, MMPs lower,MBP & PLP higher, GFAP and cytokines lower, and IAIPs higher in thebrain of IAIP—than placebo-treated ischemic fetuses. Results of thesestudies will give us some indication of mechanisms by which IAIPs exerttheir neuroprotective effects allowing for a larger proposal focusedupon IAIPs' mechanisms of action.

Alternative Procedures.

We would like to determine oligodendrocyte lineage in the placebo- andIAIP-treated ischemic brain. If the above results suggest IAIPsattenuate WM damage, we will consider this approach. In addition, if wefind higher IAIP levels in the brains of IAIP-treated fetuses, we mayseek to determine the mechanism by which IAIPs gain access to the brain.

Specific Methods:

ELISA IL-6 & IL-1β and IAIP. IL-6, IL-1β and IAIP protein concentrationswill be measured in blood by ELISA.⁹⁹ IAIP concentrations will bemeasured by a competitive ELISA (ProThera Biologics). Purified ovineIAIP will be immobilized on 96-well microplates. Rabbit anti-IAIP (R-16)will be added to samples and incubated on the well for 1 h at RT. Afterwashing, secondary HRP-conjugated anti-rabbit Ig (Invitrogen) will beincubated for 1 h. One-step TMB will be used as a substrate and colorchanges measured on spectrometer. The IAIP concentrations in samples arecalculated against a known IAIP standard.

Example 9

Perinatal hypoxic-ischemic injury (HI) is the leading cause of mortalityand long-term neurologic morbidity in premature and term infants withpregnancy and/or birth complications.¹⁻³ Although HI may be acute orchronic, affected children often develop long-term cognitive andbehavioral deficits.⁴⁻¹¹ Moreover, severity of injury and pathologicaloutcome are dependent upon sex, with increased incidence and more severelong-term deficits in male than in female infants.¹²⁻¹⁸ The mechanism(s)of these sex differences are largely unknown; however, recent dataindicate sex differences in cell death and extent of tissue damage afterHI.¹⁹⁻²⁴ These differences suggest that neuroprotective strategiesshould be tailored differentially by sex for maximal benefit. We seek toincrease knowledge of the mechanism(s) of sex differences in HI, and todetermine the comparative effects of IAIPs in sex-related differences ofHI.

To determine mechanism(s) of sex differences in HI injury andneuroprotection, we will examine pro-inflammatory cytokine expression(known to cause and/or accentuate brain injury)²⁵⁻²⁷ and thedifferential neuroprotective effects of IAIP, which is known todown-regulate cytokines in a sepsis model.²⁸ Mechanistic determinationsinclude molecular, immunological, immunohistological, and long-termbehavioral outcomes in male and female rats after HI. Our data show IAIPdepletion after ischemia in fetal sheep brain²⁹ and IAIP treatment to bethe most neuroprotective strategy examined to date in neonatal malerodents. However, given evidence of sex differences in mechanisms of andlong-term outcomes after H1, the expression of endogenous IAIP maydiffer between the sexes after HI. Through histological and long-termbehavioral study of both male and female rats, our studies examine sexdifferences in cytokine and IAIP expression after HI, and determine themodulation of key differences in cell death by this excitingneuroprotective agent. IAIP is poised to enter clinical trials forsepsis and related molecules suppress preterm labor,³⁰⁻³² and thus thistherapeutic agent could rapidly enter clinical use to attenuate HIinjury in infants. We predict a sexually dimorphic response due to keymodulators involved in sex-specific mechanisms of H-related cell death.

We propose experiments to determine IAIP and cytokine concentrations inserum and brain of male and female rats at 2, 4, 6, 8, 24 h and 7 dafter HI injury. Given increased damage and deficits for males, wehypothesize that serum and brain tissue cytokine levels are higher, andIAIP depletion greater, in male rats than female rats after HI.

We also propose to examine the neuroprotective effects of exogenous IAIPtreatment given immediately and 24 h after HI using histological andlong-term behavioral measures to determine the mechanism(s) by whichIAIP is neuroprotective. We hypothesize that IAIP treatment (30mg/kg×2)²⁵ decreases neuronal death and microglia activation in ratsexposed to HI, and that treatment is more efficacious in male thanfemale rats. The acute effects of HI and IAIP treatment can be measuredwith Fluoro Jade B (FJB; dying neurons) and EDI (activated microglia),along with IAIP modulation of sex related mechanism(s) of cell death,including caspase 3 and poly (ADP-ribose) polymerase 1 (Parp1) activity,Long-term behavioral outcome can be evaluated by Morris Water Mazeperformance (Aim 2b).

Background:

HI is a major cause of infant brain injury with an occurrence of2-4/1000 full term and 5-6/1000 premature infants,³³⁻³⁴ though incidenceand outcome appear dependent upon sex. The clinical origin of thisdifference derives from increased rates of vascular/neurologiccomplications (i.e., intra-cranial bleeds, HI complications of delivery)in male versus female neonates, as well as superior cognitive recoveryby females following HI injuries of comparable severity.³⁵ Although thecause(s) of these sex dependent differences are largely unknown, recentevidence suggests cell death mechanisms differ between the sexes,indicating the cascade of detrimental events induced by HI may differfor males and females.¹⁹⁻²⁴ Moreover, these findings suggest male andfemale neonates would likely respond differently to neuroprotectivestrategies. Although research on sex differences in incidence andoutcome of adult stroke is rapidly expanding, there is a paucity ofinformation related to this important aspect of brain injury inneonates. Therefore, addressing the importance of sex in response toearly HI injury is important.

Though the majority of neonatal HI research has almost exclusivelystudied male subjects, recent data demonstrate sex differences inintrinsic cell death pathways¹⁹⁻²⁴ as well as influences of the hormonalmilieu on the response to HI injury.³⁶⁻³⁸ Specifically, data indicatedifferences in the proportional activation of two apoptotic pathways(caspase-independent and -dependent) following HI.^(19,39) Male HI micedisplay increased Parp1 (an enzyme essential to the caspase-independentpathway) relative to females, while caspase-3 (active in thecaspase-dependent pathway) is present in higher concentration in HIfemale mice than male.²⁰ Likewise, Parp-1 knockout benefits HI males,but not females,²¹ while caspase inhibition is neuroprotective to HIfemale (but not male) animals.^(22,23) These studies emphasize theimportance of expanding our understanding of the cause of increaseddeficits and poorer outcomes in males-both in infants^(12-18, 35) and inanimal models.^(24, 38) Given these apparent differences, it is likelysex-related differences will also be observed in underlying molecularand cellular features of cell death and tissue damage after HI. Notably,pro-inflammatory cytokines are an important component of the cascadethat causes and/or accentuates brain injury,²⁵⁻²⁷ while IAIP has beenshown to effectively down-regulate cytokines in neonatal and adultsepsis models.²⁸ However, the potential sex-related differences intiming of cytokine activation (a certain contributor to injury),particularly with reference to utilization of endogenous IAIP levels inthe brain is not known, nor has it been studied between the sexes. Thus,we seek to compare potential differences in brain cytokine and IAIPconcentrations both in control and HI exposed male and female animals.

Cytokines are elevated intravascularly and in cord blood of infants wholater developed cerebral palsy,⁴⁰ in amniotic fluid of premature infantswho later developed white matter lesions,⁴¹ and in white matter frominfants who died of periventricular leukomalacia,⁴² the foremostpredictor of cerebral palsy in human infants.⁴³⁻⁴⁴ Furthermore, IAIP wasshown to increase survival in a neonatal rodent model of sepsis, in partby down regulating pro- and up regulating anti-inflammatory cytokines.²⁸Additionally, pretreatment with IAIP subunit, bikunin (purified fromurine and having a short 3-10 min half-life), is neuroprotective againststroke injury in adult rats²⁶ and attenuates white matter damage in anadult model of autoimmune encephalomyelitis (EAE).⁴⁵ In contrast, theIAIP used here (isolated from blood and with a significantly longerhalf-life, 8-12 h) represents the native complex form of the protein andis therefore feasibly a more effective neuroprotective agent.

IAIP treatment may attenuate or prevent brain damage in infants with HIby reducing pro- and enhancing anti-inflammatory cytokines. In fact,decreased IAIP has been shown to accurately predict the development ofsepsis in premature infants, whereas excreted IAIP-related fragmentssuggest these proteins are rapidly consumed during sepsis.⁴⁶Importantly, sepsis is a significant predictor of brain damage inpremature infants.⁴⁷⁻⁴⁸ Moreover, increased IAIP levels in healthy adultmale plasma (in relation to female) suggest males may require increasedIAIP due to increased sensitivity to inflammation.⁴⁹ Additionally, Parp1plays a crucial role in systemic inflammatory shock⁵⁰ and has been shownto enhance expression of pro-inflammatory mediators in models ofsepsis,⁵¹ thus indicating IAIP treatment may preferentially protectmales given their predominant use of Parp1-mediated cell death pathway.However, this response remains to be determined in neonates of bothsexes. Examination of the comparative benefits of IAIP administrationand mechanism in male and female rats after HI injury is also important.

We seek to increase understanding of sex-related differences in themechanisms of neonatal HI; and, for the first time, examine the effectof a neuroprotective agent, IAIP, in neonatal rodents of both sexesusing histological, immunological, and long-term behavioral measures.Pro-inflammatory cytokines have not been quantified, nor have theputative neuroprotective effects of exogenous IAIP treatment beencompared in the brains of male and female rats after HI. We seek todetermine why the sexes respond differently to neonatal HI, and willdetermine some of the underlying mechanisms for these differences.Finally, our data may suggest sex differences in cellular functioningafter early brain injury, indicating the importance of attention to sexin the development of optimal neuroprotective therapies for neonates.

Sex differences in long-term behavioral outcome after HI: Our data onthe long-term behavioral outcome of male and female rats with P7 HI showsignificant deficits in a modified prepulse inhibition paradigm for maleHI animals only.^(24,38) Deficits were elicited in female HI animalsonly when treated with early testosterone³⁸ or embelin,²⁴ an inhibitorof X-linked inhibitor of apoptosis (XIAP). IAIPs are known to blockapoptosis by binding to caspases in the caspase-dependent, predominatelyfemale activated pathway. Treatment preventing this endogenousinhibition results in HI-related damage and deficits in females only. Itis likely sex differences also exist in pro-inflammatory cytokines inresponse to HI and because Parp1 has been shown to mediate inflammatoryresponses, we predict therapeutic IAIP intervention to vary by sex.

IAIP Expression in the Ovine Fetal Brain: Effects of Ischemia.

Our data suggests age-dependent expression of IAIP, as plasmaconcentrations are higher in adult and newborn sheep than fetuses at 70%and 90% gestation.⁵³ Data also show substantial ischemia-induceddecreases in IAIP 4 h after ischemia in the ovine fetus as measured byWestern blot (FIG. 3, inset)²⁹ obtained using a specific polyclonalantibody against human IAIP that cross-reacts with non-human IAIP,including sheep and rat IAIP. The rapid decrease in IAIP duringischemia-reperfusion could be due to consumption of cortical IAIP,suggesting that IAIP is utilized/broken down during ischemia. Thisphenomenon raises the interesting possibility that IAIP acts as anendogenous anti-inflammatory molecule and that treatment with exogenousIAIP after ischemia could be an effective neuroprotective strategy. Thisconcept is also supported by evidence of neuroprotection by the shortacting subunit, bikunin, in EAE.⁴⁵

Neuroprotective Effects of IAIP in Neonatal Male Rats.

Our exciting preliminary data demonstrates substantial neuroprotectiveeffects of IAIP treatment for male rats with neonatal HI.⁵² Dataindicate considerable neuroprotective properties of IAIP as exogenoustreatment after ischemic surgery, but before hypoxia, inhibits loss ofcortical tissue measured by total brain weight (FIG. 2) and dramaticallyreduces the number of dying cerebral cortical neurons measured by FJBstaining (FIG. 1). IAIP treatment occurs after HI induction to show thetranslational relevance of our model to treatment in humans.

Kinetics of Intraperitoneal Administration of IAIP in Healthy NewbornMice:

We performed preliminary absorption and kinetic studies of i.p. (30 or60 mg/kg) purified human IAIP in P6 mouse pups. Five animals were usedper IAIP dose and each animal per group sacrificed at 1, 3, 6, 12, and21 h after injection. Trunk blood collected individually was analyzedfor IAIP concentration using a competitive in house ELISA developed byYPL. Monoclonal antibody 69.31, specific against the light chain ofhuman IAIP, detects systemic human IAIP in the mouse. IAIP levels peaked6 h after injection in both groups and decreased by 24 h (FIG. 12).There was no difference in the level of IAIP in the two groups,suggesting a limitation of IAIP absorption in mice. Nevertheless,results suggest that more than one IAIP injection may be needed toachieve optimal neuroprotective effects and that 30 mg/kg is anappropriate dose for our studies. Human IAIP is to be used and detectionof IAIP with this ELISA assay will be similar in neonatal rats.

Hypoxic-Ischemic Insult:

Time-mated Wistar rats will be used. On P1 litters will be culled to 5males and 5 females to reduced variability in nutrition and maternalcare between litters. On P7, HI pups will be anesthetized withisoflurane (2% induction, 1% maintenance), an incision made along themidline of the neck (approximately 0.5 cm), and the right common carotidartery exposed and cauterized. The incision will be sutured and pupsallowed to recover. Sham animals will receive incision only. Allprocedures will be done on a heating pad and temperature closelymonitored. Pups will be individually marked for identification byfootpad ink injections. After the litter has recovered, pups will returnto the dam for feeding (˜2 h). HI subjects will then be placed in anairtight chamber, through which humidified 8% oxygen will flow for 120m. A heating pad under the chamber provides warmth and pup temperaturewill be monitored. Sham animals will be placed in an identicalcontainer, without a lid or reduced oxygen flow, for 120 m. Uponcompletion, the entire litter will be returned to the dam. CAH hasroutinely performed this procedure with excellent survivability.

Methods:

Both HI and sham procedures, as well as sacrificing times after HI/shamprocedure, will be balanced within litter (Table 3). Because HI resultsin extensive tissue damage to the ipsilateral hemisphere, an increasednumber of HI animals are needed for sufficient tissue for analysis. Theduration of survival after HI is based upon our data showing greatestdecrease in IAIP 4 h after ischemia.²⁹ Because we seek to determine thedetailed time course of the pattern of cytokine and IAIP expressionafter HI, brains will be harvested at the above specified time intervalsby rapid decapitation and snap frozen. Trunk blood will be collected forplasma assays.

TABLE 3 Male Sham Female Sham Male HI Female HI 2 h n = 6 n = 6 n = 10 n= 10 4 h n = 6 n = 6 n = 10 n = 10 6 h n = 6 n = 6 n = 10 n = 10 8 h n =6 n = 6 n = 10 n = 10 24 h  n = 6 n = 6 n = 10 n = 10 7 d n = 6 n = 6 n= 10 n = 10

Tissue Processing:

Frozen cortical samples extracted from each brain hemisphere will bepooled with brain tissue from like-treated, same sex animals for IAIPanalyses by Western immunoblot. Trunk blood from like-treated, same sexanimals will be collected and pooled for ELISA analyses for plasma IAIPand cytokine concentrations.

Western Immunoblot:

IAIP determination in tissue: Aliquots for equal protein loading (50μg/well) will be fractionated using 4-12% BIS TRIS SDS-polyacrylamidegel (Invitrogen) electrophoresis and immunoblotted onto PVDF membrane(Polyvinylidene difluoride, 0.2 micron, Bio-Rad Laboratories) using asemi-dry technique. Immunoblots will be blocked with a solution of 10%milk and 90% Tris-buffered saline with 0.1% Tween-20 solution (TBST) forone h at room temperature (RT), washed three times in TBST for 10m/wash, and probed overnight with primary 1:5000 rabbit polyclonalprimary antibody (ProThera Biologics) at 4′C. Next, immunoblots will bewashed three times with TBST for 10 m/wash, and incubated for one h with1:10000 goat anti-rabbit horseradish peroxidase conjugated secondaryantibody (Alpha Diagnostic) at RT. After four washes in TBST at 10m/wash, immunoblots will be developed using enhanced chemiluminescencesolution (ECL Prime, Western Blotting Detection Reagents, AmershamPharmacia Biotech, Inc.) before exposure to autoradiography film(Phoenix Research Products). Molecular weight standards (Bio-RadLaboratories) will be included in each immunoblot. A human IAIP standardwill be used as a positive control for all immunoblots to establish withcertainty that proteins identified in rat tissue are the same as theknown proteins. IAIP bands (125 and 250 kDa) intensities will beanalyzed with a Gel-Pro Analyzer (Media Cybernetics). The experimentaldensitometry values will be normalized to beta actin. Group samples willbe analyzed on at least two Western immunoblots.

Caspase-3 Determination in Tissue:

Protein concentration will be determined using the methods published byWhitaker and Granum⁵⁴, adapted for microplates.²⁰ Pro-caspase 3 ismeasured at 32 kDa and cleaved caspase 3 measured at 29 kDa.

Parp-1 Determination in Tissue:

Formation of PAR polymers via nuclear protein modification is a markerof Parp1 activity and will be measured by Western blot using rabbitanti-PAR polyclonal antibody LP96-10 (Biomol, SA-276).²¹

ELISA Assay:

IAIP determination in plasma: Purified rat IAIP will be immobilized on amicroplate at RT for 1 h. Wells will be blocked with 5% non-fat driedmilk for 1 h then washed with PBS+0.05% Tween 20 (PBS-T). Samples willbe diluted in PBS and rabbit polyclonal antibody against IAIP (R-16)added and incubated for 45 m. IAIP present in the sample will competewith the immobilized IAIP on the plate for antibody binding. Afterwashes with PBS-T, HRP-conjugated goat-anti rabbit IgG will be added andincubated for 30 m. Bound antibodies will be visualized by addingOne-Step TMB Substrate Solution and color change will be read using a650 nm filter on a spectrometer. IAIP levels in samples will becalculated against the standard curve included in the assay using anIAIP solution with known concentration.

Cytokine Determination in Tissue:

Protein concentrations of IL-6, IL-10, TNF-α, and IL-1β will be measuredin brain samples by ELISA. Samples will be transferred to 10 ml conicalmicro tubes and combined with homogenization buffer consisting of 20 mMTris-HCL, pH 7.4; 2.0M NaCl; 1 mM EDTA; 1 mM EGTA; 0.5% Deoxycholate; 1%Igepal; proteinase inhibitor cocktail (1 mM PMSF and 1 mg/ml of each ofthe following, Aprotinin, Leupeptin, Pepstatin A). After sonication for1 min and centrifugation at 14,000 rpm for 30 min at 4° C., the totalprotein content of the supernatants will be determined with an assay kit(Pierce, Rockford, Ill.). The IL-6, IL-10, TNF-α, and IL-1β content ofsupernatants will also be performed by ELISA. An ELISA scanner (ThermoFisher Scientific) will be used to measure the optical density of thetotal protein at 562 nm, and IL-6, and IL-1β at 450 nm. Antibodies torat IL-6, IL-10, IL-1β, and TNF-α against different epitopes areavailable and will be used in cytokine-specific ELISA's.

Cytokine Determination in Plasma:

Quantikine ELISA kits for IL-6, IL-10, TNF-α, and IL-1β are available(R&D Systems) and will be used for plasma cytokine analysis.

Analysis:

Results will be expressed as mean±SD. Multivariate analysis of variance(ANOVA) will be used to determine the effect of HI on IAIP expressionand cytokine concentration in the ipsilateral (injured) versus thecontralateral cerebral cortices, where the specific factors areIpsilateral/Contralateral, Treatment (sham/HI), Sex (male/female) andTime after HI (2, 4, 6, 8, 24 h, 7 d). If a significant difference isdetected by ANOVA, the Fischer least significant difference test willdetermine differences among the hemispheres, time after HI, and sexdifferences among the different groups. Further, the relationshipbetween IAIP expression and cytokine concentration after HI will beexamined by correlational analysis with dummy coding variables to adjustfor the different time periods. P<0.05 will be considered statisticallysignificant.

Investigation of the Putative Neuroprotective Effects of Exogenous IAIPThrough Histological Measures.

Methods:

I.P. injection of 30 mg/kg IAIP in 100 μL NaCl solution or 100 μL NaClvehicle will be given immediately after induced HI (i.e., upon releasefrom the hypoxic chamber; see Table 4), and 24 h later. This dose andschedule was selected based on preliminary data indicatingneuroprotection in male rats undergoing HI, ELISA determinations inmice, and previous studies showing improved survival rates aftersystemic infection.²⁵ The current design is proposed with translation toclinical practice in mind and IAIP is therefore being given afterinjury. At 72 h after HI, all subjects will be overdosed withpentobarbital and perfused with 5 ml cold (4° C.) PBS followed by 5 ml4% paraformaldehyde. A72 h endpoint was chosen to ensure assessment ofthe inflammatory response to injury (often delayed compared to themolecular events relating to necrosis (6 h) and apoptosis (24 h).

TABLE 4 Male Female Sham Vehicle n = 10 n = 10 Sham IAIP n = 10 n = 10HI Vehicle n = 10 n = 10 HI IAIP n = 10 n = 10

Histological Processing and Stereological Assessment:

Serial sections will be cut using a vibrating microtome. Every fifthslice will be mounted and labeled for generalized neuronal cell deathusing FJB, while every sixth slice will be labeled for activatedmicroglia using EDI. Processed tissue will be digitized and visualizedusing a Zeiss AxioImager M2 microscope system with color camera, remotestage, and Windows-based PC using StereoInvestigator software(Burlington, Vt.). Whole numbers of degenerating neurons and activatedmicroglia (from cerebral cortex, thalamus, hippocampus, and basalganglia) will be estimated, blind to treatment, using the OpticalFractionator probe in StereoInvestigator.

Statistical Analysis:

A multivariate ANOVA will be used to assess the differences betweentotal number of dying cells and total number of activated microgliaacross brain regions between experimental conditions and across sex.Simple effects analyses for each sex will involve one-way ANOVAs forindividual brain regions for both counts. These analyses will reveal anysubtle differences in cell death markers or microglia activation acrossgroups.

Investigation of the Putative Neuroprotective Effects of Exogenous IAIPThrough Long-Term Behavioral Measures

We also seek to investigate the potential long-term neuroprotectiveeffects of IAIP treatment on behavioral performance. The Morris WaterMaze (MWM) task requires the identification of a submerged platformusing spatial (extra-maze) cues and examines learning and spatialreference memory. Rats will undergo insult and IAIP treatment. Animalswill be housed with dams until weaning at P21, pair housed until P50,and single housed in adulthood. Animal weight will be recorded daily asan initial measure of IAIP side effects.

Water Escape (P34):

Each rat will be released at the end of an oval tub and required to swimto the opposite end to a visible escape platform. Rats will be guided tothe platform if they have not located it after 45 s, and will remainthere for 10 s. This procedure is used to assess any group differencesin baseline motor behavior.

Morris Water Maze (P35-39):

The maze consists of a round tub with a submerged platform in a fixedlocation and extra-maze cues (shapes painted on walls, the doorframe,etc.) Each rat will enter at one of four start positions and will swimuntil finding the hidden platform (45 s max). It will return to its cageunder a warming lamp for 2-3 min before the next trial. On the remainingtrials, the rat will enter the maze from one of the remaining startpoints. This procedure will be repeated over 5 days.

Probe Trial (P39):

After conclusion of Day 5 trials, the platform will be removed and ratswill enter the tub at a random location. Time spent swimming in thequadrant that previously contained the submerged platform, as well ascrossings made in the area that previously contained the platform willbe measured.

Data Acquisition and Analysis:

Ethovision XT video tracking system (Naldus) will be used to recordbehavior of rats in the maze. Detailed recording of distance traveledand time to reach the platform for individual animals will be evaluatedby repeated measure ANOVAs. Variables include Sex (male/female),Treatment (HI/Sham), Drug (IAIP/vehicle), Time to reach the platform,Distance traveled, and Day. For probe trials, time spent in the correctquadrant and the number of crossings in the area the platform waspreviously located will also be measured. One-way ANOVAs will be usedfor simple effects analyses for each day of testing and for comparisonof probe trial data between groups.

Possible Outcomes and Alternative Procedures:

Our studies are the first to measure IAIP and cytokine concentrations,and the effect of IAIP treatment, in both sexes after Ill. Though theprobability of IAIP levels remaining stable at the measured time pointsis unlikely, we recognize we may not find differences in levels betweenthe sexes. Nonetheless, we still predict identifying neuroprotectiveeffects of IAIP treatment based upon recent studies^(26, 45) and ourdata in male rats only. Importantly, the development of neuroprotectantsnonspecific to sex is possible if similar expression and/or depletion ofIAIP is shown in males and females. Additionally, we recognizeendogenous levels of IAIP may differ between the sexes in a timedependent manner and therefore optimal intervention with IAIP may differin timing for males and females. However, IAIP will be given immediatelyafter HI based on our data, but also so that these important proteinsare ‘on board’ as soon as would be possible in the clinical setting(i.e., if the timing of the HI event was known). The opportunity forstudies exploring treatment at the time point of lowest endogenouslevels, or the efficacy of treatment at later time points are importantconsiderations for future studies. Secondly, we expect a positiveoutcome in both histological and behavioral measures after IAIPadministration. If attenuation of injury is not observed (i.e., reducedFJB and EBI staining) in either sex, the dose, number of doses, ortiming of doses may be modified. Moreover, exogenous IAIP treatment maybe more efficacious to one sex, requiring differential dosing/treatmentstrategies to achieve equivalent efficacy. Finally, hypothermia iscurrently the only approved therapy for the treatment of HI in humanneonates. The use of IAIP as a neuroprotective agent may be enhanced ifcombined with hypothermia treatment.

Ethical Considerations:

Our use of a live animal model is essential to investigate the acutecellular and molecular mechanisms of HI with no alternatives in theproposed research. Careful evaluation or all protocols has ensuredanimals will be closely monitored for the duration of study. We performaseptic surgery with utmost consideration for comfort and post-surgicaltrauma is minimal. Technical difficulties are unlikely as all techniqueshave been previously employed by the investigator and are used routinelyin our laboratory and no animals are requested for techniquedevelopment. A rat model is ideal due to prior successful studiesperformed by CAH and tissue availability for protein/assay analysis.Also, this is the most practical study design for this proposal due toshorter gestation and increased offspring per litter than sheep.

Impact of Study:

Considering the tremendous amount of research surrounding sexdifferences in adult stroke—and the tremendous advancements that havecome from this research—it is difficult to understand the paucity ofresearch surrounding similar issues in the neonate. Males and femalesdiffer in behavioral, physiological, and genetic levels (even inneonates), so it is not surprising that sex differences manifest inepidemiology and pathogenesis of HI. Our research shows considerablesignificance in the following areas: 1) understanding how and why malesand females respond differently to HI injury during the neonatal period;2) examining mechanisms underlying these differences, specifically withregard to the role of cytokines and IAIP; and 3) understanding howexogenous IAIP treatment may modulate both anatomical and behavioralindices of injury. The results of our studies move the field of sexdifferences in HI injury forward and contribute to the clinicalimplementation of sex-specific neuroprotectants for infants sufferingfrom HI injury.

OTHER EMBODIMENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth.

All patents patent applications and publications mentioned herein areincorporated by reference to the same extent as if each independentpatent or patent application was specifically and individually indicatedto be incorporated by reference in its entirety.

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1. A method of treating, reducing, or inhibiting ischemia or a conditionresulting from ischemia, a viral infection, cancer metastasis, or astroke or a condition resulting from stroke in a patient in need thereofcomprising administering to said patient a composition comprisinginter-alpha inhibitor (IaI) and/or pre-alpha inhibitor (PaI). 2.(canceled)
 3. The method of claim 1, wherein said ischemia is hypoxicischemia. 4-7. (canceled)
 8. The method of claim 1, wherein saidischemia is acute ischemia. 9-10. (canceled)
 11. The method of claim 1,wherein said method comprises reducing the severity of said ischemia orsaid condition resulting from ischemia.
 12. (canceled)
 13. The method ofclaim 1, wherein said ischemia results from a medical condition, atraumatic injury, or a congenital malformation.
 14. The method of claim13, wherein said ischemia results from ischemic hemorrhagic stroke. 15.The method of claim 1, wherein said ischemia occurs in a tissue or celltype selected from skeletal muscle, smooth muscle, cardiac muscle,connective tissue, mesenchymal tissue, gastrointestinal tissue,placenta, liver, heart, kidney, intestine, lung, colon, kidney, bladder,testes, skin, bone, brain, cerebral cortex, choroid plexus, cerebrum,cerebellum, neurons, astrocytes, and meningeal cells. 16-20. (canceled)21. The method of claim 1, wherein said method comprises reducing theseverity of said ischemia or said condition resulting from ischemia insaid patient. 22-27. (canceled)
 28. The method of claim 1, wherein saidpatient is a human. 29-30. (canceled)
 31. The method of claim 1, whereinsaid composition is administered at a dosage of 1 mg/kg body weight to50 mg/kg body weight.
 32. The method of claim 1, wherein saidcomposition is administered at a dosage ranging from 50 mg/dose to 1000mg/dose.
 33. (canceled)
 34. The method of claim 1, wherein saidcomposition comprises a pharmaceutically acceptable excipient, diluent,or carrier.
 35. The method of claim 34, wherein said composition is asolid or a liquid. 36-37. (canceled)
 38. The method of claim 34, whereinsaid composition is formulated for inhalation, insufflation,nebulization, injection, oral, rectal, topical, or intraperitonealadministration, intracerebral injection, intravenous delivery, orinfusion. 39-46. (canceled)
 47. A method of providing neuroprotection toa patient in need thereof, said method comprising administering to saidpatient a composition comprising inter-alpha inhibitor (IaI) and/orpre-alpha inhibitor (PaI).
 48. A method of treating a wound in a patientin need thereof, said method comprising administering to said patient acomposition comprising inter-alpha inhibitor (IaI) and/or pre-alphainhibitor (PaI). 49-50. (canceled)
 51. The method of claim 1, whereinsaid viral infection is influenza, Dengue fever, or West Nile fever.52-53. (canceled)
 54. The method of claim 1, wherein the patient is anadult.
 55. The method of claim 13, wherein: i) said medical condition isselected from peripheral artery disease, type 1 or type 2 diabetes,atherosclerotic cardiovascular disease, intermittent claudication,critical limb ischemic disease, stroke, cancer, myocardial infarction,inflammatory bowel disease, carotid occlusion, umbilical cord occlusion,low birth-weight, premature birth, pulmonary insufficiency, peripheralneuropathy, and bleeding, ii) said traumatic injury is selected fromwound, burn, laceration, contusion, bone fracture, infection, andsurgical procedure, or iii) said congenital malformation is selectedfrom hernia, cardiac defect, and gastrointestinal defect.
 56. The methodof claim 48, wherein the composition comprises polyethylene glycol orsodium carboxymethylcellulose and is administered topically.